ApoE antibody, fusion protein, and its use

Abstract

To provide methods and compositions for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment and / or neurodegeneration using antibodies, peptides, fusion proteins, or genome editing systems that modulate HSPG / heparin binding affinities of ApoE.SOLUTION: Provided is an Fc fusion protein comprising an Fc region of an antibody fused to a polypeptide comprising an HSPG-binding domain of wild-type ApoE or mutant ApoE, the HSPG-binding domain comprising a specific amino acid sequence.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 853,67 filed on May 28, 2019 No. 6, and U.S. Provisional Patent Application No. 62 / 873,019 filed on July 11, 2019 The entire contents of the foregoing are incorporated herein by reference.

[0002] Federally Sponsored Research or Development This invention was made with Government support under Grant No. OD019833 awarded by the National Institutes of Health, Grant No. AG054671, AG031581, and A G19610 awarded by the National Institute on Aging, and Grant No. N S100121 and NS110048 awarded by the National Institute of Neurolo gical Disorders and Stroke. The Government has certain rights in this invention.

[0003] Methods and compositions for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment by modulating the heparan sulfate proteoglycan (HSPG) / glycosaminoglycan (GAG ) heparin binding affinity of apolipoprotein E (ApoE) ) are described herein.

Background Art

[0004] Alzheimer's disease (AD) is a chronic neurodegenerative disease that usually starts slowly and gradually worsens over time. It is the cause of 60 to 70% of dementia cases. The disease course ​​​​​The progression is related to plaques and neurofibrillary tangles in the brain. Some are temporary symptoms. While the condition can be improved, there is currently no treatment to stop or reverse its progression. The accumulation, aggregation, and deposition of amyloid-beta (Aβ) peptides in Alzheimer's disease ( ApoE plays a central role in the formation of AD lesions. ApoE regulates Aβ aggregation and metabolism, thereby contributing to A It has been demonstrated by increasing evidence that it strongly influences the formation of lesion D (Fu et al. ., Mol Neurodegener 11:37, 2016). ApoE is involved in amyloidogenesis, aggregation, and cranial cell production. It affects Alanth, is a component of amyloid plaques, and exacerbates tau-mediated neurodegeneration. ApoE is 299 amino acids long and has only one amino acid at positions 112 and 158. Alternatively, three major alleles distinct from each other by two amino acids (epsilon 2, ipsilon Silon 3 and Epsilon 4): ApoE2 (cys112, cys158), Apo E3(cys112, arg158), and ApoE4(arg112, arg158 It is a polymorphism that has ). Therefore, AD and cognitive impairment associated with dementia or mild cognitive impairment. Targeting and regulating the function of ApoE protein to treat or prevent cognitive decline. There is a need for therapeutic substances. [Overview of the project]

[0005] In one embodiment, this disclosure relates to wild-type or mutant apolipoprotein E (ApoE) One or more (e.g., 1, 2, 3, or 4) HSPG binding sites, or HS One or more (e.g., 1, 2, 3, or 4) allosteric modulations of PG binding It is characterized by isolated monoclonal antibodies that specifically bind to the site. Several implementations In this state, the antibody is TEELRVRLASHLRK (SEQ ID NO: 3) and at least 9 5% (e.g., 96%, 97%, 98%, 99%, or 100%) identical amino acid sequences It binds to a polypeptide having TEEL. In some embodiments, the antibody is TEEL RVSLASHLRK (SEQ ID NO: 2) and at least 95% (e.g., 96%, 97%, 9%) It binds to polypeptides having the same amino acid sequence (8%, 99%, or 100%). In some embodiments, the antibody is wild-type or mutant ApoE2, ApoE3 , or one or more (e.g., 1, 2, 3, or 4) HSPG bindings of ApoE4 It connects to the body part.

[0006] In some embodiments, the antibody has a heavy chain variable region (VH) and a light chain variable region (V Competing with and / or binding to the same epitope as the reference anti-ApoE antibody containing L), the same reference The VH and VL of the antibody (i) have the amino acid sequences shown in SEQ ID NO: 13 and (ii) The amino acid sequence shown in SEQ ID NO: 12; (ii) The amino acid sequence shown in SEQ ID NO: 23 (iii) The amino acid sequence and the amino acid sequence shown in SEQ ID NO 22; (iii) SEQ ID NO 3, respectively. The amino acid sequence shown in 3 and the amino acid sequence shown in SEQ ID NO: 32; or (iv) The amino acid sequences shown in SEQ ID NO: 43 and SEQ ID NO: 42, respectively. Includes sequence. In some embodiments of any of the antibodies described herein, anti The body competes with the same epitopes as the reference anti-ApoE antibody, including heavy and light chains, and / or The heavy and light chains of the reference antibody bind to the amino acid sequence shown in SEQ ID NO: 53 and It contains the amino acid sequence shown in SEQ ID NO: 52.

[0007] In another aspect, VH includes VHCDR1, VHCDR2, and VHCDR3, Rabini VL containing VLCDR1, VLCDR2, and VLCDR3, anti-ApoE anti The body is VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 , and VLCDR3 are (i) sequence numbers 7, 8, 9, 4, 5, and 6, respectively; (ii) (iii) Each of the following, sequence numbers 17, 18, 19, 14, 15, 16; (iii) Each of the following, sequence number Sequence numbers 27, 28, 29, 24, 25, 26; (iv) Sequence numbers 37, 38, 39 respectively. , 34, 35, 36; or (v) respectively, sequence numbers 47, 48, 49, 44, 45, 46, including. In some embodiments of any of the antibodies described herein, ( i) VH and VL have different amino acid sequences from those shown in SEQ ID NOs. 13 and 12, respectively. At the very least, 75%, 80%, 85%, 90%, 95%, or 100% of the amino acids are identical. (ii) VH and VL are shown in SEQ ID NOs. 23 and 22, respectively. The amino acid sequence and at least 75%, 80%, 85%, 90%, 95%, or 100% (iii) VH and VL each contain identical amino acid sequences; (iii) VH and VL are, respectively, sequence number 3 The amino acid sequences shown in 3 and 32 and at least 75%, 80%, 85%, 90%, 9 Contains 5% or 100% identical amino acid sequences; or (iv) VH and VL However, each of them is at least 75% of the amino acid sequences shown in SEQ ID NOs. 43 and 42, and 8 Contains amino acid sequences that are 0%, 85%, 90%, 95%, or 100% identical. In some embodiments of any of the antibodies described in the document, the antibody is SEQ ID NO: 53 and the amino acid sequences shown in 52 and at least 70%, 75%, 80%, 85%, 90% It contains heavy and light chains with amino acid sequences that are %, 95%, or 100% identical, respectively. In some embodiments, the antibodies are mouse IgG1, IgG2a, IgG2b, The antibody contains IgG2c or IgG3 heavy chain constant region. In some embodiments, the antibody is , containing human IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions. In embodiments, the antibody includes a human kappa or human lambda light chain constant region. In the embodiment, the antibody is a whole antibody, a single-domain antibody, a humanized antibody, a chimeric antibody, or a two-domain antibody. Heavily specific antibodies, Fv, scFv, sc(Fv)2, diabody, nanobody, Fab, Or F(ab')2. In some embodiments, the antibody has an extended half-life. The antibody further includes a portion. In some embodiments, the antibody further includes a blood-brain barrier permeable portion. It includes. In some embodiments, the antibody further includes a detectable label. In the embodiments thereof, a pharmaceutical composition comprising any of the antibodies described herein is Provided in book. In some embodiments, any of the antibodies described herein Polynucleotides encoding these are provided herein. In some embodiments, Vectors containing polynucleotides as described in the specification are provided herein. In embodiments thereof, polynucleotides described herein or Host cells containing a vector are provided herein. In another embodiment, an anti-ApoE antibody A method for producing (a) any of the host cells described herein, the antibody (b) a step of culturing under conditions that enable the expression of the antibody, and (b) a step of isolating the antibody. Methods including a pu are provided herein. In some embodiments, the method is used to a human The process further includes the step of formulating an antibody as a sterile preparation suitable for administration.

[0008] In another embodiment, STEELRVRLASHLRKLRKRLLRDADDLQK( Sequence ID 57), STEELRVSLASHLRKLRKRLLRDADDLQK (array Number 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (array) Number 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (Sequence ID 60) is at least 95% identical to an amino acid sequence selected from the group consisting of the above. Includes the HSPG-binding domain of wild-type ApoE or mutant ApoE, which contains the noacid sequence. Fc fusion proteins are provided herein. In some embodiments, Fc fusion The protein contains the Fc region of the human antibody. In some embodiments, the human antibody is It is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 molecules. In several embodiments, the Fc fusion protein comprises one of the Fc fusion proteins described herein. Pharmaceutical compositions are provided herein. In some embodiments, as described herein... Polynucleotides encoding Fc fusion proteins are provided herein. In that embodiment, a vector comprising any of the polynucleotides described herein. —is provided herein. In some embodiments, the polen described herein Host cells containing a creotide or a vector described herein are provided herein. .

[0009] In another embodiment, (i) STEELRVRLASHLRKLRKRLLRDADDL QK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (Sequence ID 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (Sequence ID 59), and RLVQYRGEVQAMLGQSTEELRVSLASHL At least 95% identical to an amino acid sequence selected from the group consisting of RKL (SEQ ID NO: 60). The HSPG-binding domain of wild-type ApoE or mutant ApoE, containing the amino acid sequence of the following: , and (ii) a pharmaceutically acceptable adjuvant for inducing an immune response Pharmaceutical compositions are provided herein.

[0010] In another embodiment, any of the antibodies described herein or the antibodies described herein A pharmaceutical composition comprising human cells expressing any of the Fc fusion proteins is provided herein. It will be done.

[0011] In another embodiment, an antibody, Fc fusion protein, or pharmaceutical combination described herein A human being who needs a therapeutically effective dose of any of the products, including the step of administering a therapeutically effective dose of one of the products to a human subject. In the subjects, cognitive decline associated with dementia and / or mild cognitive impairment and / or This refers to methods that improve, slow down, delay, prevent, or reverse neurodegeneration. The law is provided herein.

[0012] In another embodiment, in a human subject in need, dementia and / or mild dementia To improve, slow down, or reduce the onset of cognitive decline and / or neurodegeneration associated with intellectual disability. A method for delaying, preventing, or reversing the APOE gene, wherein the method involves (i) APOE inheritance. Encodes a gRNA molecule containing a target domain derived from the offspring and a targeting domain complementary to it. (ii) a viral vector containing a nucleotide sequence; (ii) a nucleotide encoding the Cas9 molecule (iii) a viral vector containing an Otid sequence; and (iii) the position 19 of the APOE gene Instead of cytosine, adenine:g.45412013C>A is included in the template nucleic acid. The step includes administering a rusvector to the subject, wherein the administration results in the subject The generation of one or more ApoE R136S alleles in one or more cells Methods for producing this are provided herein. In some embodiments, the gRNA molecule The facilitating domain is the same as the sequence from Table 7, or differs by 3 nucleotides or less. The bottom row contains a different array.

[0013] As used herein, “prevent” means to reduce the risk of developing a disorder. It means...

[0014] In some embodiments, the human subject is diagnosed with Alzheimer's disease, and There is a risk of developing it. In some embodiments, the human subject is APOE4 It has one or more copies of the allele. In some embodiments, the human subject is At least one gene selected from the group consisting of APP, PSEN1, and PSEN2 The offspring has one or more mutations. In some embodiments, the human subject is One or more mutations in additional genes that cause autosomal dominant Alzheimer's disease Different (for example, see Bateman et al., Alzheimer's Research & Therapy 3(1): 1, 2011). (having been done) In some embodiments, the human subject has the third of chromosome 21. It has all or part of a copy of the brain. In some embodiments, the human subject is brain imaging Established biomarkers such as images, or those obtained from blood or CSF samples. He is diagnosed with Alzheimer's disease. In some embodiments, human subjects are Over 50 years old (for example, over 55, over 60, over 65, over 70) (Above, above 75, above 80, above 85, above 90, or above 95) .

[0015] In some embodiments, human subjects are diagnosed with a disorder selected from the group consisting of the following: Those who have or are at risk of developing: vascular cognitive impairment, vascular dementia, cortical dementia Autosomal overt cerebral artery disease with inferior infarction and leukoencephalopathy (CADASIL), subcortical infarction and Autosomal occult cerebral artery disease with leukoencephalopathy (CARASIL), Lewy body dementia, frontal lobe dementia Temporal lobe dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease Neurodegenerative diseases, cerebrovascular diseases, brain injuries, chronic traumatic brain injury, tauopathy, amyloidosis Creutzfeldt-Jakob disease, synukleinopathy, retinal degeneration, glaucoma, retinal damage optic nerve degeneration, and aging.

[0016] In yet another aspect, the development of an early-onset neurodegenerative disease involves the following steps: A method for identifying a human subject is provided herein: a step of obtaining a biological sample from the subject. The presence of at least one variant allele of APOE3 in the biological sample, or A step of detecting the presence of a mutant ApoE3 gene product in the biological sample; and a step of detecting the presence of a mutant ApoE3 gene product in the biological sample. Early-onset neurodegenerative diseases are caused by the presence of a different APOE3 allele or gene product. A step to identify subjects that are less likely to develop symptoms. In some embodiments, the biological sample is blood. These include cerebrospinal fluid, saliva, urine, tears, vitreous fluid, aqueous humor, or tissue samples. Several procedures are performed. In terms of morphology, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, retinal degeneration, or glaucoma. In some embodiments, retinal Membrane degeneration is age-related macular degeneration. In some embodiments, the step of detection is against This includes determining the sequence of the APOE3 allele in elephants. In some embodiments, The step to detect the mutation at R136 compared to the wild-type ApoE3 protein is to compare it with the wild-type ApoE3 protein. To determine the presence or absence of an APOE3 sequence encoding an ApoE3 protein containing the ApoE3 protein. This includes the following. In some embodiments, the mutation in R136 is R136S, R It is 136H or R136C. It is used to treat early-onset neurodegenerative diseases as described herein. In some embodiments of any method for identifying human subjects less susceptible to the disease, the method is: Steps to select subjects for inclusion in clinical trials, and optionally, experimental procedures The step to be performed, or if the subject does not have a mutant APOE3 allele, the subject Further steps include excluding from clinical trials the early-onset neurological disorders described herein. In some embodiments of a method for identifying human subjects less likely to develop sexually transmitted diseases The method includes the step of selecting subjects for inclusion in a clinical trial, and optionally, In the step of performing experimental treatment, or if the subject has a mutant APOE3 allele, the subject This further includes a step to exclude them from clinical trials.

[0017] Unless otherwise specified, all technical and scientific terms used herein are those of the art. It has the same meaning as commonly understood by the present invention. The methods and materials used in this invention For the purposes described herein; other suitable methods and materials known in the art It can also be used. The materials, methods, and examples are illustrative only and do not limit the scope of the material. Not intended to be used in connection with all publications, patent applications, patents, arrangements, and other documents mentioned herein. Database entries and other references are incorporated herein by reference as a whole. In case of any conflict, this specification, including its definitions, shall prevail.

[0018] Other features and advantages of the present invention are described in the following detailed description and figures, as well as in the claims. The scope should make it clear. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows a structural model of the wild-type APOE3 protein. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are shown. The amino acid positions for the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are also shown. [Figure 2] This figure shows representative Sanger sequencing results for APOE from control, proband, and offspring samples. [Figure 3] This diagram shows the family tree of the subject, with circles representing females, squares representing males, diamonds representing individuals whose gender is concealed for privacy reasons, arrowheads depicting proband individuals with MCI, and shading indicating individuals with a history of dementia. Deceased individuals are marked with crossed bars. Individual APOE and PSEN1 genotypes are appropriately indicated to protect anonymity. [Figure 4-1]Figures 4A and 4B show fundus photographs of the right and left eyes, respectively. Figure 4C shows an infrared image of the right eye depicting a cross-section of the retina (line) as seen in Figure 4D. Figure 4D shows the results of optical coherence tomography (OCT) of the right eye. Figure 4E shows an infrared image of the left eye. Figure 4F shows the results of OCT imaging of the left eye. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] This figure shows brain imaging results indicating the amount of amyloid plaques and PHF tau in the brains of PSEN1 mutation carriers with late-onset MCI (mild cognitive impairment) and PSEN1 mutation carriers who developed MCI at the typical age for this mutation (44 years). [Figure 6] This figure shows measured levels of amyloid, tau, hippocampal volume, and glucose metabolism in PSEN1 E280A mutation carriers. Red dots represent measurements for the carriers with two APOE3ch alleles and exceptionally late onset of MCI. Black dots represent PSEN1 E280A mutation carriers with MCI who were at a younger age than typical for their family at the time of MCI onset. Gray dots represent PSEN1 E280A mutation carriers who have not yet developed MCI. [Figure 7] This figure shows the rate of Aβ42 fiber formation detected by thioflavin T fluorescence in the presence of wild-type APOE3, APOE3ch, or in the absence of APOE. The change in relative fluorescence units (RFU) is plotted against time in minutes (min) (***P<0.001, ****P<0.0001). [Figure 8] This figure shows a schematic of split cyferase complementation induced by amyloid oligomerization (top), and the percentage of luminescence obtained after 24 hours in culture medium from 293T cells transfected with ApoE3ch or ApoE3 wild-type in a split cyferase complementation assay. [Figure 9] This figure shows the ELISA results for heparin binding affinity of APOE2 and APOE4. [Figure 10]This figure shows the Western blot analysis of heparin binding affinity for ApoE2, ApoE3, ApoE4, and ApoE3ch. [Figure 11-1] This figure shows the ELISA results for heparin binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12] Figure 12A is a schematic diagram showing the experimental setup for testing the specificity of a monoclonal ApoE3 antibody in blocking ApoE3 / heparin binding. Figures 12B and 12C are schematic diagrams showing the process of passing the monoclonal antibody and pre-incubated ApoE3 protein through a heparin-conjugated column, followed by washing and elution. [Figure 13] This figure shows the results from BCA assays performed on various fractions from a heparin-conjugated column. Figure 13A shows the amount of ApoE3 in various fractions in the absence of ApoE3 antibody. Figure 13B shows the amount of ApoE3 in various fractions in the presence of ApoE3 antibody. [Figure 14] This figure shows Western blot results indicating the amount of ApoE3 in various fractions with and without pre-incubation with monoclonal ApoE3 antibody. [Figure 15-1] This figure shows the ELISA analysis results for 19G10-2, 23B2, 2H79-1, 30E1-2, 16H8, 25F1-2, and 29G10-1 antibodies. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 15-3] This is a continuation of Figure 15-1. [Figure 15-4] This is a continuation of Figure 15-1. [Figure 15-5] This is a continuation of Figure 15-1. [Figure 15-6] This is a continuation of Figure 15-1. [Figure 15-7] This is a continuation of Figure 15-1. [Figure 16]This figure shows the Western blot analysis of heparin-binding affinity of ApoE3 treated with wild-type ApoE3 peptide and ApoE3ch mutant peptide. [Figure 17] This figure shows a model of the interaction between ApoE fragments and heparin. Figure 17A shows a model of a wild-type ApoE fragment containing amino acids 129-157 that interact with heparin. Figure 17B shows a model of the ApoE R136S fragment containing amino acids 129-157 that interact with heparin. Figure 17C shows a model of a wild-type ApoE fragment containing amino acids 114-144 that interact with heparin. Figure 17D shows a model of the ApoE R136S fragment containing amino acids 114-144 that interact with heparin. [Figure 18-1] This figure shows the heparin affinity chromatography and Western blot analysis of antibody 1H4. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19] This figure shows the ELISA results of 1H4-2 serum tested using ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D). [Figure 20] This figure shows the ELISA results of 1H4-2 serum tested using ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, and ApoE3ch peptide. [Figure 21] This figure shows representative ELISA profiles of serial dilutions of antibody 1H4 incubated with either human recombinant ApoE3 or mouse recombinant ApoE3. [Figure 22] This figure shows the ELISA results for monoclonal 1H4 antibodies purified from cloned hybridomas. [Figure 23-1] This figure shows the heparin affinity chromatography and Western blot analysis of antibody 7C11. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24] This figure shows the ELISA results from testing 7C11-1 serum using ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D). [Figure 25] This figure shows the ELISA results from testing 7C11-1 serum with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, or ApoE3ch peptide. [Figure 26] This figure shows the ELISA results for monoclonal 7C11-1 antibody purified from cloned hybridomas. [Figure 27] This figure shows the results of ELISA screening of the heparin-binding domain of recombinant proteins of ApoE3 wild-type (WT) and ApoE3ch mutants using 19G10-2 antibodies. [Figure 28-1] This figure shows the heparin affinity chromatography and Western blot analysis of antibody 19G10-2. [Figure 28-2] This is a continuation of Figure 28-1. [Figure 29] This figure shows Western blotting of ApoE3 WT incubated with 19G10-2 serum antibody. [Figure 30] This figure shows a representative ELISA illustrating the difference in binding between serum 19G10-2 and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3 WT or ApoE3ch. [Figure 31] This figure shows an enlarged view of the Y-axis in Figure 30. [Figure 32] This figure shows the ELISA results for monoclonal 19G10-2 antibody purified from cloned hybridomas. [Figure 33] This figure shows the results of ELISA screening of the heparin-binding domain of recombinant proteins of ApoE3 wild-type (WT) and ApoE3ch mutants using 25F1-2 antibodies. [Figure 34-1]This figure shows the heparin affinity chromatography and Western blot analysis of antibody 25F1-2. [Figure 34-2] This is a continuation of Figure 34-1. [Figure 35] This figure shows Western blotting of ApoE3 WT incubated with 25F1-2 monoclonal antibody. [Figure 36] This figure shows a representative ELISA illustrating the difference in binding between 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3 WT or ApoE3ch. [Figure 37] This is a view of the Y-axis magnified in Figure 36. [Figure 38] This figure shows the ELISA results for monoclonal 25F1-2 antibody purified from cloned hybridomas. [Figure 39] This figure shows the ELISA screening of 1343 antibodies against the heparin-binding domain of recombinant ApoE3 wild-type (WT) and ApoE3ch mutant proteins. [Figure 40] This figure shows the ELISA screening of 1343 antibodies against the heparin-binding domain of recombinant ApoE3 wild-type (WT) and ApoE3ch mutant proteins. [Figure 41-1] Figure 41A shows the Western blot analysis of ApoE in the protein fraction eluted from a heparin column in and out of the presence of antibody 1343. Figure 41B shows the ELISA analysis of the said fraction. [Figure 41-2] This is a continuation of Figure 41-1. [Figure 42] Figure 42A shows an exemplary experimental overview of an intraocular model of inducible ApoE-dependent tau hyperphosphorylation. Figure 42B shows PHF tau in a control retina injected with PBS. Figure 42C shows a retina injected with recombinant human ApoE3. [Figure 43-1]This figure shows PHF tau in a control retina compared to a retina injected with either mouse 1H4-2 antibody or humanized 1343Ah antibody. [Figure 43-2] This is a continuation of Figure 43-1. [Figure 43-3] This is a continuation of Figure 43-1. [Figure 44] This figure shows representative binding measurements of the ApoE3 protein to 1H4 at increasing concentrations (nM) in a protein A biosensor. [Modes for carrying out the invention]

[0020] This disclosure describes homozygosity of APOE3ch (APOE3 Christchur Having two copies of the ch(R136S) mutation contributes to the clinical onset of Alzheimer's disease. It is associated with significant resistance to heparan sulfate proteoglobulin, and the R136S mutation is associated with heparan sulfate proteoglobulin We discovered that lycan (HSPG) significantly reduces the ability of ApoE to bind heparin. Therefore, this disclosure contains wild-type ApoE and / or the R136S mutation. Antibodies that bind to ApoE isoforms (e.g., ApoE and HSPG / GAG / H) Antibodies that block interactions between parin and / or reduce binding between them. To do so. Peptide fragments of the wild type and mutant ApoE containing the R136S mutation (for example, Fusion proteins containing HSPG / GAG / heparin-binding domains are also being considered. These proteins can be administered via human cells expressing such compositions. This disclosure further relates to a small molecule that blocks the interaction between ApoE and HSPG / heparin. This relates to methods for screening small molecules of the ApoE locus in genome editing. Compositions and methods for editing using a collection system are also provided. Antibodies described herein, Fusion proteins, small molecules, and genome editing systems are used in dementia and / or mild cognitive impairment. Cognitive impairment and / or neurodegeneration associated with mild cognitive impairment (MCI), e.g., Alzheimer's disease. - Diseases, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, and The antibodies described herein are useful in the treatment or prevention of Huntington's disease. Synthetic proteins, small molecules, and genome editing systems are also used in neurodegenerative diseases and cerebrovascular conditions. Useful in the treatment or prevention of brain injury, retinal degeneration, optic nerve degeneration, or retinal damage. .

[0021] Apolipoprotein E (ApoE) Apolipoprotein E variants are major contributors to susceptibility to late-type AD in AD. It is a genetic modifier. The APOE4 allele is derived from cysteine ​​at position 112. This leads to a change to ginine, and in single-allelic carriers, it is associated with a five-fold increase in the risk of Alzheimer's disease (AD). In homozygous carriers, the increase reaches 20 times. APOE2 is located at position 158. This causes an amino acid change from arginine to cysteine, which is protective against AD. The APOE3 allele is considered neutral (Corder et al., Nat Genet (7) 180) -184, 1994;Hauser et al., Cure Alzheimer Res (10); 808-817, 2013). ApoE is It affects amyloid production, aggregation, and clearance, and is a component of amyloid plaques. It exacerbates tau-mediated neurodegeneration. The APOE allele also affects lipid metabolism and cardiovascular health. Control tube risk. Approximately 5-10% of APOE2 homozygous individuals have hyperlipoproteinemia I Type II (HLP III) develops, but other rare variants of APOE are autosomal. It is associated with overt HLP III. HLP III is associated with plasma cholesterol and Due to increased triglyceride levels, as well as the presence of nodular or linear palmar xanthomas It is characterized as follows: The APOE allele modifies AD risk and causes HLP III. The mechanism is not fully understood. The prominent APOE properties are affected by specific mutations. 1) Binding affinity to lipids and LDL receptors; 2) Its N-terminal domain (Am The nature of interdomain interactions between the no acids (1-199) and the C-terminal domain (216-299) Quality; 3) Differences in the ability to form homo-oligomers mediated by the C-terminal domain (Frieden et al., PNAS (109):8913-8918, 2012; Georgiadou et al., PLoS) One (6)e27037, 2011; Lalazar et al., J Biol Chem (263)3542-3545, 1988).

[0022] Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. Two or three HS chains are in extremely close proximity to the cell surface or extracellular matrix proteins. It exists as an attached proteoglycan (HSPG). The HSPG portion is in its original form. It is present in hundreds of proteins located in the sacral membrane and extracellular matrix. More mediated protein-protein interactions include amyloid and tau pathologies. Heparan sulfate plays a crucial role in numerous processes related to the pathogenesis of Luzheimer's disease. It is a member of the glycosaminoglycan family of carbohydrates, and is structurally non-heparin. They are always closely related. Both consist of variable sulfated repeating disaccharide units. Heparan sulfate binds to numerous extracellular proteins. Collectively, these bind to "heparin." These are often called "interactomes" or "heparin-binding proteins," and they In affinity chromatography of the related polysaccharide, heparin... This is because it becomes more isolated.

[0023] Exemplary amino acid sequence of human ApoE3 protein (Uniprot accession number) The code P02649 is shown below:

[0024] [ka]

[0025] At least two HSPG / heparin-binding domains were identified in human ApoE. It is located in the N-terminal domain and the C-terminal domain (Weisgraber et al. al. J Biol Chem, 261(5):2068-76, 1986;Saito et al., J Biol Chem, 278(17):14782- 7, 2003). HSPG / heparin-binding domain near arginine 136 (R136) The N-terminal HSPG / heparin-binding domain is used in full-length lipid-depleted and defatted ApoE. It can function in that, but the HSPG / heparin-binding domain in the C-terminal domain is N It can only function in the absence of terminal domains and in degreased ApoE. N-terminal HSP The G / heparin-binding domain is well characterized, specifically amino acid residue 14 of SEQ ID NO: 1. Includes 2-147 (bold). The C-terminal HSPG / heparin-binding domain is not very sufficient. It is not characterized by lysine (K) at position 233 of Sequence ID 1, and amino acids This includes residues 211-218 and 243-272, and contains other charged amino acids nearby. The inventors found that arginine at position 136 of ApoE is involved in the heparin binding of ApoE. It shows that it plays an important role. I don't intend to be bound by theory, but it is possible The mechanism is allosteric, mediated by arginine at position 136, heparin binding. It is a regulation. Allosteric regulation as used herein is an allosteric regulation Allosteric It is involved in the regulation of ligand binding through the binding of regulatory factors. In some embodiments In this context, one of the allosteric modifications regarding the binding of ApoE to HSPG / heparin is also If multiple sites are shown in Sequence ID No. 1, then the arginine at position 136 of ApoE Contains nin. "HSPG / heparin-binding domain", "HSPG / heparin-binding site" The terms "HSPG-binding domain" and "HSPG-binding site" are interchangeable in this specification. It can be used.

[0026] Anti-ApoE antibody Anti-Ap that binds to wild-type or mutant ApoE protein (human ApoE protein) oE antibodies are provided. In some cases, the antibodies described herein are wild-type ApoE antibodies. It binds to proteins (e.g., ApoE2, ApoE3, or ApoE4), but mutant It does not bind to ApoE proteins (e.g., ApoEch). In some cases, as specified herein The antibodies described bind to the mutant ApoE protein (e.g., ApoEch), but , wild-type ApoE protein (e.g., ApoE2, ApoE3, or ApoE4) and It does not bind. In some cases, the antibodies described herein do not bind to the mutant ApoE protein. (e.g., ApoEch) and wild-type ApoE protein (e.g., ApoE2, ApoE It binds to both (3) or (ApoE4).

[0027] In some cases, the antibodies provided herein are wild-type ApoE proteins (e.g., A Blocks the interaction between poE2, ApoE3, or ApoE4 and HSPG. The antibodies provided herein are ApoE proteins (e.g., ApoE2, ApoE3, Alternatively, the binding affinity of ApoE4) to HSPG may be reduced or modified. In this context, the antibodies provided herein are HSPG-binding hosts for wild-type ApoE protein. It binds to HSPG / ApoE. In some cases, the antibodies provided herein are HSPG / ApoE conjugated. One or more sites of allosteric regulation (e.g., amino acid position 136 of ApoE) ) binds to. In some cases, the antibodies described herein contribute to fibrillation and / or It reduces myloid oligomerization.

[0028] In some cases, the antibodies provided herein are TEELRVSLASHLRK (sequence number) The amino acid sequence in wild-type or mutant ApoE, comprising or consisting of (2) and It binds. In some cases, the antibodies provided herein are TEELRVRLASHLRK (SEQ ID NO: 3) Amino acids in wild-type or mutant ApoE, comprising or consisting thereof It binds to the acid sequence. In some cases, the amino acid sequence TEELRVSLASHLRK(sequence number) No. 2) comprises or consists of an epitope for the antibody provided herein. In this case, the amino acid sequence TEELRVRLASHLRK (SEQ ID NO: 3) is used in this specification. The provided antibody contains or consists of an epitope. Varian of these sequences. For example, these sequences are at least 80%, 85%, 90%, or 95% identical. Those can also be used.

[0029] The calculation of "identity" between two arrays can be performed as follows: The arrays are... Align for appropriate comparison (for example, first and for optimal alignment) A gap can be introduced in one or both of the second nucleic acid sequences for the purpose of comparison. (This allows for the ignoring of non-identical sequences). Alignment is performed for comparison purposes. The length of the array should be at least 70% (e.g., at least 80%, 90%) of the length of the reference array. , or 100%). Then the nucleotide at the corresponding nucleotide position Compare. The nucleo whose position in the first sequence is the same as the corresponding position in the second sequence. If occupied by cydos, those molecules are identical at their positions. Between two sequences Percent identity needs to be introduced for optimal alignment of the two sequences. Shared by their array, taking into account the number of gaps and the length of each gap. It is a function of the number of identical positions.

[0030] The comparison of arrays and the determination of percentage identity between two arrays are performed using mathematical algorithms. This can be achieved by... Percent identity is measured using the Blossum 62 matrix and the PAM250 matrix. Needleman and using either the NWSgapdna.CMP matrix. Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (GCG software) It is incorporated into the GAP program in the package (available at gcg.com) Determined using ( ). In some embodiments, two amino acids or nucleos Percent identity between cyd sequences is determined by the PAM120 weighted residue table, 12 gap length penalties. Luthi and E. Meyers and W. Mill, using a gap penalty of 4. er ((1989) CABIOS, 4:11-17) algorithm (ALIGN program (version 2) It can be determined using the (which is incorporated into .0)).

[0031] The term "antibody" in this disclosure refers to the whole antibody (minibody, nanobody, or antibody). (As an antonym for body fragment), bispecific antibody, tetravalent antibody, multispecific antibody, minibody, nano It is intended to cover bodies and antibody fragments. In some cases, the antibodies in this disclosure ApoE antibodies are total antibodies. In some cases, the anti-ApoE antibodies in this disclosure are chimeric, human. , or a humanized antibody. In some cases, the heavy chain constant region of an anti-ApoE antibody is human I This is the constant region of IgG1, human IgG2, human IgG3, or human IgG4. In this case, the light chain steady region is the human kappa steady region. In other cases, the light chain steady region is This is the tramda constant region. In some cases, the antibody of this disclosure has low effector function. Designed to do so (e.g., by Fc modifications such as N297Q, T299A, etc.). (See reference) (Incorporated herein by Wang, X., Mathieu, M. & Brezski, RJ Protein Ce) (See also ll (2018) 9: 63. doi.org / 10.1007 / s13238-017-0473-8). In some cases, anti The Fc parts of the body are hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, and hIgG4 Fc, hIgG1agly Fc, hIgG2 SAA Fc, hIgG4(S228 P) Fc, or hIgG4(S228P) / G1 agly Fc((effector (Minimizing functionality) In this configuration, the CH1 and CH2 domains are "fixed" It is an IgG4 with a hinge (S228P) and is aglycosylated. CH3 dome The gene is hIgG1 or hIgG4(S228P) IgG1. In one example, the antibody had reduced effector function, and the following three scaffolds... It has one of the following: hIgG1 agly(N297Q); hIgG2 SAA(Vafa et See al. Methods, 65(1):114-26 (2014); and hIgG4P / G1 agly (USA) (See Patent Application Publication No. 2012 / 0100140).

[0032] In some embodiments, the antibody or its ApoE-conjugated fragment described herein The antibodies 1H4-2, 7C11-1, and 19G10- are illustrated in the following Examples section. 2, 23B2(1343), 2H79-1, 30E1-2, 16H8, 25F1-2, o The binding properties and / or biological properties of 29G10-1 are outlined as shown. It exhibits the characteristic properties.

[0033] In some embodiments, the present disclosure relates to binding with wild-type human ApoE or a portion thereof. The present invention provides an antibody having one or more of the following properties: (i) wild-type human Apo (ii) It binds to E and KD ≤ 20 nM with high affinity; (ii) In binding to heparin, wild-type heparin (iii) Competes with ApoE; and (iii) Paired spiral fibers (PHFs) in retinal cells It reduces the formation of cavities.

[0034] In some embodiments, this disclosure relates to mutant human ApoE (e.g., ApoEch (and others) those having a mutation at amino acid position 136 of human ApoE, or a part thereof. The present invention provides an antibody that binds to a molecule and has one or more of the following properties: (i) mutant antibody ApoE (for example, ApoEch, etc., at amino acid position 136 of human ApoE) (ii) Binds to mutants with high affinity KD ≤ 20 nM; (ii) Binding to heparin (iii) competes with wild-type human ApoE in (iii) and (iii) paired helical in retinal cells It reduces fibrous (PHF) tau formation.

[0035] None of the anti-ApoE antibodies described herein are used in the treatment of dementia or mild cognitive impairment (MC). I) Disorders related to (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, Frontotemporal dementia, Parkinson's disease, or Huntington's disease), neurodegenerative diseases, cerebrovascular diseases It is useful in treating or preventing sexually transmitted diseases, brain injuries, retinal degeneration, or retinal damage.

[0036] Exemplary antibody 1H4-2 Antibody 1H4-2, APOE: KLH-CTEELRVRLASHLRK-CONH It was produced by immunization with 2. The complementarity-determining region (CDR) and heavy chain of 1H4-2. The amino acid sequences of the variable region and the light chain variable region are provided below.

[0037] [Table 1]

[0038] Variable light chain: Nucleotide sequence: Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0039] [ka]

[0040] Amino acid sequence: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0041] [ka]

[0042] Variable heavy chain: Nucleotide sequence Signal sequence -FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0043] [ka]

[0044] Amino acid sequence: Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0045] [ka]

[0046] In some cases, the anti-ApoE antibody contains three VH CDRs of antibody 1H4-2. It includes VLs, including 3 VL CDRs. The 6 CDRs are non-exclusively Kabat, C hothia, enhanced Chothia, contact, IMGT, or This can be based on any definition known in the art, such as Honegger's definition. These CDRs are, for example, from the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key This can be determined by using _annotation.cgi.

[0047] In one example, the anti-ApoE antibody of this disclosure (i) has the amino acid combination shown in SEQ ID NO: 7 VHCDR1 containing the column, VHCDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and VH containing VHCDR3 containing the amino acid sequence shown in SEQ ID NO: 9; and (ii) VLCDR1 containing the amino acid sequence shown in column number 4, and the amino acid sequence shown in sequence number 5 VLCDR2 containing the column and VLCDR3 containing the amino acid sequence shown in SEQ ID NO: 6 Includes VL.

[0048] In some cases, the anti-ApoE antibody has the amino acid sequence shown in SEQ ID NO: 13 and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Contains VH which is 97%, 98%, or 99% identical. In some cases, the anti-ApoE antibody is , at least 75%, 80%, 85%, 90% of the amino acid sequence shown in Sequence ID No. 12, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical It contains VL, which is one example of an anti-ApoE antibody, which contains the amino acids shown in SEQ ID NO: 13. The amino acid sequence shown in VH and SEQ ID NO: 12 is at least 85% identical to the sequence. It contains VL, which is at least 85% identical. In another example, the anti-ApoE antibody in SEQ ID NO: 13 The amino acid sequences shown are at least 90% identical to those shown in VH and SEQ ID NO: 12. It contains VL which is at least 90% identical to the amino acid sequence. In yet another example, anti-ApoE The antibody is VH, which has the same amino acid sequence as shown in SEQ ID NO: 13, and SEQ ID NO: 12. It contains VL, which is identical to the amino acid sequence.

[0049] In some cases, the antibody of this disclosure that binds to ApoE is shown in SEQ ID NO: 13. Reference Intakes include VH having an amino acid sequence and VL having the amino acid sequence shown in SEQ ID NO 12. This antibody competes with or binds to the same epitope as the illuminating antibody.

[0050] Example antibody 7C11-1 Antibody 7C11-1, APOE: KLH-CTEELRVRLASHLRK-CON It was produced by immunization with H2 (SEQ ID NO: 54). Complementary determination region of 7C11-1 ( The amino acid sequences of the CDR, heavy chain variable region, and light chain variable region are provided below. .

[0051] [Table 2]

[0052] Variable heavy chain: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0053] [ka]

[0054] Amino acid sequence: Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0055] [ka]

[0056] Variable light chain: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0057] [ka]

[0058] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0059] [ka]

[0060] In some cases, the anti-ApoE antibody contains three VH CDRs of antibody 7C11-1. and includes VL containing 3 VL CDRs. 6 CDRs are non-limited Kabat, Chothia, enhanced Chothia, contact, IMGT, also This can be based on any definition known in the art, such as Honegger's definition. These CDRs are, for example, from the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / ke This can be determined by using y_annotation.cgi.

[0061] In one example, the anti-ApoE antibody of this disclosure is (i) an amino acid shown in SEQ ID NO: 17 VHCDR1 containing the sequence, VHCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, VH containing VHCDR3 containing the amino acid sequence shown in SEQ ID NO: 19; and (i i) VLCDR1 containing the amino acid sequence shown in SEQ ID NO: 14, and SEQ ID NO: 15 VLCDR2 containing the amino acid sequence, and V containing the amino acid sequence shown in SEQ ID NO: 16 Includes VL, including LCDR3.

[0062] In some cases, the anti-ApoE antibody has an amino acid sequence that is at least as shown in SEQ ID NO: 23. 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Contains VH which is 97%, 98%, or 99% identical. In some cases, the anti-ApoE antibody is , at least 75%, 80%, 85%, 90% of the amino acid sequence shown in Sequence ID No. 22, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical It contains VL, which is one example of an anti-ApoE antibody, which is the amino acid shown in SEQ ID NO: 23. The amino acid sequence shown in VH and SEQ ID NO: 22 is at least 85% identical to the sequence. It contains VL, which is at least 85% identical. In another example, the anti-ApoE antibody in SEQ ID NO: 23 The amino acid sequences shown are at least 90% identical to those shown in VH and SEQ ID NO: 22. comprises a VL that is at least 90% identical to the amino acid sequence. In yet another example, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 23 and the amino acid sequence set forth in SEQ ID NO: 22 comprises a VL that is identical to the amino acid sequence set forth therein.

[0063] In some cases, the antibodies of this disclosure that bind ApoE are antibodies that compete for or bind to the same epitope as a reference antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 23 and a VL having the amino acid sequence set forth in SEQ ID NO: 22. a VH having the amino acid sequence and a VL having the amino acid sequence set forth in SEQ ID NO: 22, the reference antibody that competes for or binds to the same epitope as the reference antibody.

[0064] Exemplary Antibody 19G10-2 Antibody 19G10-2 was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2 The complementarity determining regions (CDRs) and the amino acid sequences of the heavy chain variable region and the light chain variable region of 19G10-2 are provided below. The amino acid sequences of the heavy chain variable region and the light chain variable region are provided below.

[0065] [Table 3]

[0066] Variable light chain: Nucleotide sequence: Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0067] [Chemical Formula]

[0068] Amino acid sequence: Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0069]

Chemical Formula

[0070] Variable Heavy Chain Analysis: Nucleotide Sequence Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0071]

Chemical Formula

[0072] Amino Acid Sequence: Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0073]

Chemical Formula

[0074] In some cases, the anti-ApoE antibody comprises a V comprising the three VH CDRs of antibody 19G10-2 H and a VL comprising the three VL CDRs. The six CDRs include, but are not limited to, Kabat , Chothia, enhanced Chothia, contact, IMGT, or may be based on any definition known in the art, such as the Honegger definition . These CDRs can be determined, for example, by using the AbYsis database (bioinf.org.u k / abysis / sequence_input / key_annotation / k ey_annotation.cgi).

[0075] In one example, the anti-ApoE antibody of this disclosure comprises (i) the amino acid set forth in SEQ ID NO: 27 VHCDR1 comprising the sequence, VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH comprising VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; and (i i) VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, the amino acid sequence set forth in SEQ ID NO: 25 VLCDR2 comprising the amino acid sequence, and VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 comprising the VL comprising LCDR3.

[0076] In some cases, the anti-ApoE antibody is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 33 (75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical VH. In some cases, the anti-ApoE antibody is at least 75%, 80%, 85%, 90%, identical to the amino acid sequence set forth in SEQ ID NO: 32 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical VL. In one example, the anti-ApoE antibody has an amino acid sequence set forth in SEQ ID NO: 33 a VH that is at least 85% identical to the sequence and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 32 comprising a VL that is at least 85% identical. In another example, the anti-ApoE antibody is directed to SEQ ID NO: 33 a VH that is at least 90% identical to the amino acid sequence set forth and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 32 comprising a VL that is at least 90% identical to the amino acid sequence. In yet another example, the anti-ApoE the antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 32 comprising a VL that is identical to the amino acid sequence set forth.

[0077] In some cases, an antibody of this disclosure that binds ApoE comprises a VH having the amino acid sequence set forth in SEQ ID NO: 33 a reference VH having the amino acid sequence and a VL having the amino acid sequence set forth in SEQ ID NO: 32 This antibody competes with or binds to the same epitope as the illuminating antibody.

[0078] Exemplary antibody 25F1-2 Immunizing antibody 25F1-2 with KLH-CTEELRVSLASHLRK-CONH2 It was fabricated by doing so. The complementarity determination region (CDR) and heavy chain variable region of 25F1-2 The amino acid sequences of the variable region of the light chain are provided below.

[0079] [Table 4]

[0080] Variable light chain analysis: Nucleotide sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0081] [ka]

[0082] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0083] [ka]

[0084] Variable heavy chain analysis: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0085] [ka]

[0086] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0087] [ka]

[0088] In some cases, the anti-ApoE antibody contains three VH CDRs of antibody 25F1-2. and includes VL containing 3 VL CDRs. 6 CDRs are non-limited Kabat, Chothia, enhanced Chothia, contact, IMGT, also This can be based on any definition known in the art, such as Honegger's definition. These CDRs are, for example, from the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / ke This can be determined by using y_annotation.cgi.

[0089] In one example, the anti-ApoE antibody of this disclosure is (i) an amino acid shown in SEQ ID NO: 37 VHCDR1 containing the sequence, VHCDR2 containing the amino acid sequence shown in SEQ ID NO: 38, VH containing VHCDR3 containing the amino acid sequence shown in SEQ ID NO: 39; and (i i) VLCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, and SEQ ID NO: 35 VLCDR2 containing the amino acid sequence, and V containing the amino acid sequence shown in SEQ ID NO: 36 Includes VL, including LCDR3.

[0090] In some cases, the anti-ApoE antibody has an amino acid sequence that is at least as shown in SEQ ID NO: 43. 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Contains VH which is 97%, 98%, or 99% identical. In some cases, the anti-ApoE antibody is , at least 75%, 80%, 85%, 90% of the amino acid sequence shown in Sequence ID No. 42, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical It contains VL, which is one example of an anti-ApoE antibody, which contains the amino acids shown in SEQ ID NO: 43. The amino acid sequence shown in VH and SEQ ID NO: 42 is at least 85% identical to the sequence. It contains VL, which is at least 85% identical. In another example, the anti-ApoE antibody is in sequence number 43. The amino acid sequences shown are at least 90% identical to those shown in VH and SEQ ID NO: 42. It contains VL which is at least 90% identical to the amino acid sequence. In yet another example, anti-ApoE The antibody is VH, which has the same amino acid sequence as shown in SEQ ID NO: 43, and SEQ ID NO: 42. It contains VL, which is identical to the amino acid sequence.

[0091] In some cases, the antibody of this disclosure that binds to ApoE is shown in SEQ ID NO: 43. VH has an amino acid sequence and VL has the amino acid sequence shown in SEQ ID NO 42. This antibody competes with or binds to the same epitope as the illuminating antibody.

[0092] Exemplary antibody 1343ab Immunizing antibody 1343ab with KLH-CTEELRVSLASHLRK-CONH2 This was done by creating the complementarity-determining region (CDR) and the full-length heavy and light chain mesh. The no-acid sequence is provided below.

[0093] [Table 5]

[0094] Fully long heavy chain

[0095] [ka]

[0096] Fully long light chain

[0097] [ka]

[0098] EC N-linked glycosylation was detected at 292 on the constant region N of the heavy chain. Loss of nucleotides was observed in the heavy chain.

[0099] In some cases, the anti-ApoE antibody contains three VH CDRs of antibody 1343ab. and includes VL containing 3 VL CDRs. 6 CDRs are non-limited Kabat, Chothia, enhanced Chothia, contact, IMGT, also This can be based on any definition known in the art, such as Honegger's definition. These CDRs are, for example, from the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / ke This can be determined by using y_annotation.cgi.

[0100] In one example, the anti-ApoE antibody of this disclosure is (i) an amino acid shown in SEQ ID NO: 47 VHCDR1 containing the sequence, VHCDR2 containing the amino acid sequence shown in SEQ ID NO: 48, VH containing VHCDR3 containing the amino acid sequence shown in SEQ ID NO: 49; and (i i) VLCDR1 containing the amino acid sequence shown in SEQ ID NO: 44, and SEQ ID NO: 45 VLCDR2 containing the amino acid sequence, and V containing the amino acid sequence shown in SEQ ID NO: 46 Includes VL, including LCDR3.

[0101] In some cases, the anti-ApoE antibody has the amino acid sequence shown in SEQ ID NO: 53 and at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, Weights that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical It contains a chain. In some cases, the anti-ApoE antibody has the amino acid sequence shown in SEQ ID NO: 52 and a small amount At least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical It contains a light chain, which is shown in SEQ ID NO: 53. In one example, the anti-ApoE antibody contains the amino acids shown in SEQ ID NO: 53. The heavy chain is at least 80% identical to the sequence and the amino acid sequence shown in SEQ ID NO: 52 is less identical to the sequence. It contains light chains that are at least 80% identical. In another example, the anti-ApoE antibody, SEQ ID NO: 53, The heavy chain and the sequence shown in SEQ ID NO: 52 are at least 85% identical to the amino acid sequence shown. It contains a light chain that is at least 85% identical to the amino acid sequence. In yet another example, anti-ApoE The antibody has a heavy chain identical to the amino acid sequence shown in SEQ ID NO: 53 and SEQ ID NO: 52. It contains a light chain that is identical to the amino acid sequence.

[0102] In some cases, the antibody of this disclosure that binds to ApoE is shown in SEQ ID NO: 53. The reference includes a heavy chain having an amino acid sequence and a light chain having the amino acid sequence shown in SEQ ID NO 52. This antibody competes with or binds to the same epitope as the illuminating antibody.

[0103] Chimeric, human, or humanized antibodies having any of the above antibody CDR sequences are specified in this specification. It can be prepared according to the method described in the book.

[0104] antibody fragment Antibody fragments (e.g., Fab, Fab', F(ab')2, Fabc, and Fv) are, It can be prepared by protein digestion of intact antibodies. For example, antibody fragments can be processed into papain. It can be obtained by treating the entire antibody with an enzyme such as pepsin or plasmin. Papain digestion of all antibodies produces F(ab)2 or Fab fragments; pepsin digestion of all antibodies Digestion produces F(ab')2 or Fab'; and plasmin digestion of all antibodies produces Fac This produces fragment b.

[0105] Alternatively, antibody fragments can be synthesized recombinantly. For example, the antibody of interest Nucleic acids encoding somatic fragments are constructed, introduced into expression vectors, and expressed in appropriate host cells. It can be done. For example, Co, MS et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, Methods in Enzymology, 178:476-496 (1989): Pluckth. un, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Me thods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymo logy, (1989) 121:663-669 (1989); and Bird, RE et al., TIBTECH, 9:132-137 (19 See 91). Antibody fragments are expressed in Escherichia coli (E. coli) and secreted from E. coli (E. coli). Therefore, it is possible to easily produce large quantities of these fragments. Antibody sterilization The fragments can be isolated from an antibody phage library. Alternatively, Fab'-SH fragmentation can be performed. The compound is directly recovered from E. coli and chemically coupled to F(a b') Can form 2 fragments (Carter et al., Bio / Technology, 10:163-167 (1 992)). Another approach is to directly obtain the F(ab')2 fragment from recombinant host cell culture. It can be isolated. It contains salvage receptor-binding epitope residues, which can be isolated in vivo. The Fab and F(ab)2 fragments with increased decay time are described in U.S. Patent No. 5,869,046. It is written in the book.

[0106] Conjugate antibody The antibodies disclosed herein are polymers (e.g., polyethylene glycol (PEG) PEG-modified polyethyleneimine (PEI) (PEI-PEG), polyglutamic acid (PGA)(N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer Polymeric substances such as MAR, hyaluronic acid, radioactive materials (for example, 90 Y, 131 I) Firefly It binds to a variety of molecules, including photochemicals, luminescent substances, haptens, enzymes, metal chelates, and drugs. It may be a conjugated antibody.

[0107] In some embodiments, the antibodies described herein are, for example, blood, serum, and Or, in other tissues including the brain, its stabilization and / or retention in circulation, for example at least 1.5 times, 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times or modified in a portion that enhances by 50 times. For example, the antibodies described herein are Polymers, for example, substantially non-antigenic polymers, for example, polyalkylene oxides or It can associate (e.g., conjugate) with polyethylene oxide. A suitable polymer is actually It varies qualitatively by weight. From approximately 200 Daltons to approximately 35,000 Daltons (or Molecules in the range of approximately 1,000 to 15,000 and 2,000 to 12,500 Polymers having a number-average weight may be used. For example, the antibodies described herein are water Soluble polymers, for example, hydrophilic polyvinyl polymers, for example, polyvinyl alcohol, It can be conjugated with polyvinylpyrrolidone. Examples of such polymers include poly Alkylene oxide homopolymers, for example, polyethylene glycol (PEG) or polypropylene Repropylene glycol, polyoxyethylene polyols, their copolymers, and Block copolymer (provided that the water solubility of the block copolymer is maintained) Examples include (under certain conditions). Additional useful polymers include polyoxyalkylenes, for example, Polyoxyethylene, polyoxypropylene, and polyoxyethylene and polyoxyp propylene block copolymers; polymethacrylates; carbomers; and branched or Examples include unbranched polysaccharides. In some embodiments, as described herein... The antibody has a portion that enhances its permeability across the blood-brain barrier (for example, Pardridge, J Cereb B Modified by the description in Flow Metab 32(11):1959-1972, 2012. Exemplary. The blood-brain barrier permeable portion contains glucose transporter type 1 (GLUT1), catio Monocarboxylic acid transporter type 1 (CAT1), monocarboxylic acid transporter type 1 (MCT1), concentrated nucleoside transporter type 2 (CNT2), active efflux transporter Porter (AET) (e.g., p-glycoprotein, and Pardridge, J. Cereb Blood F) Examples include those described in low Metab 32(11):1959-1972, 2012, but are not limited to these. It is not possible. Additional blood-brain barrier permeable portions are known in this art.

[0108] The above conjugate antibodies are antibodies described herein or those with a lower molecular weight. It can be prepared by chemically modifying the antibody type. The method for modifying antibodies is described in this technique. Well known in the field of technology (for example, U.S. Patent No. 5,057,313 and (and U.S. Patent No. 5,156,840).

[0109] Anti-ApoE antibodies are either in the form of the full-length (or whole) antibody of the anti-ApoE antibody, or in the form of a small molecule. Quantitative type (e.g., bioactive antibody fragment or minibody), e.g., Fab, Fab', F( It can take the forms ab')², Fv, Fd, dAb, scFv, and sc(Fv)². Other anti-ApoE antibodies included by the disclosure include single variable chains such as VH or VL. Examples include single-domain antibodies (sdAbs) containing the biologically active fragment. Moller et al., J. Biol. Chem., 285(49): 38348-38361 (2010);Harmsen et al., Appl Microbiol. Biotechnol., 77(1):13-22 (2007); U.S. Patent Application Publication No. 2005 / 0079574 See the detailed description and Davies et al. (1996) Protein Eng., 9(6):531-7. Like all antibodies, sdAb is a specific antigen (e.g., ApoE2, ApoE3, ApoE4, or Apo It can selectively bind to Ech. It has a molecular weight of only 12-15 kDa. dAb is much smaller than typical antibodies, and also contains Fab fragments and single-strand variable fragments. It's even smaller.

[0110] In a particular embodiment, an anti-ApoE antibody or its antigen-binding fragment or its lower fraction The antibody specifically binds to the HSPG / heparin-binding domain of ApoE, and the following occurs: When administered to human patients with one or more of the following conditions or to the following animal models, the severity of the symptoms Reduces: dementia and / or mild cognitive impairment (MCI) (e.g., Alzheimer's disease) Diseases, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Hunchback (Nton's disease, or those associated with neurodegeneration). In certain embodiments, anti-ApoE Antibodies or their antigen-binding fragments or their low molecular weight antibodies are used in ApoE HSPG / heparin It specifically binds to the binding domain and has one or more of the following characteristics in human patients or the following: When administered to animal models, it reduces the severity of symptoms in: neurodegenerative diseases, cerebrovascular diseases. (For example, stroke, carotid artery stenosis, vertebral artery stenosis, or aneurysm), brain injury (for example, trauma) Acquired brain injury, retinal degeneration, glaucoma, or retinal damage. Anti-ApoE antibody or These characteristics of the low molecular weight antibody are measured according to methods known in the art. It is possible.

[0111] nucleic acids, vectors, host cells This disclosure also features nucleic acids encoding antibodies disclosed herein. VH CDR1, VH CDR2, and VH CDR3 are anti-ApoE antibodies described below. Nucleic acids encoding the above are provided herein. The VL of anti-ApoE antibodies described herein Nucleic acids encoding CDR1, VL CDR2, and VL CDR3 are also provided. The anti-ApoE antibodies described herein are VH CDR1, VH CDR2, and VH CD The nucleic acids encoding R3, VL CDR1, VL CDR2, and VL CDR3 are the original Provided in detail. The heavy chain variable region (VH) of the anti-ApoE antibody described herein is coated The nucleic acid and / or the light chain variable region (VL) of the anti-ApoE antibody described herein. Nucleic acids encoding the human heavy chain constant region and / or are also provided. In some cases, the human heavy chain constant region and / or The VH of the anti-ApoE antibodies described herein, each linked to the human light chain constant region. Nucleic acids encoding and / or VL are provided herein. Anti-A described herein Nucleic acids encoding both VH and VL of poE antibodies are also provided herein. Therefore, the nucleic acids described herein include human antibodies (e.g., human IgG1, IgG2, I). Examples include nucleic acids that encode the Fc region of IgG3 or IgG4. In some cases, Nucleic acids include human antibodies that have been modified to reduce or eliminate effector function. Nucleic acids that encode the Fc region (for example, N297Q in the human IgG1 Fc region or One example is the T299A substitution (numbering using EU numbering). In some cases, Nucleic acids include hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, and hIgG4 F c, hIgG1agly Fc, hIgG2 SAA Fc, hIgG4(S228P) The Fc portion, which is Fc or hIgG4(S228P) / G1 agly Fc, is coated Nucleic acids are listed as examples.

[0112] Vectors containing any of the above nucleic acids (e.g., expression vectors) are also included herein. It will be disclosed.

[0113] Furthermore, this disclosure relates to host cells (e.g., bacterial cells) containing the above vector or nucleic acid. Regarding cells (such as yeast cells, insect cells, or mammalian cells).

[0114] Method for obtaining anti-ApoE antibodies A method for producing useful anti-ApoE antibodies in this method is also provided herein. To produce antibodies, for example, monospecific, polyclonal, or monoclonal antibodies. General methods for this are known in the art. Regarding monoclonal antibodies... The method involves either in vivo or in vitro testing of the antigen of interest (for example, Mammals that have been previously immunized with peptide antigens (such as those described herein) (e.g.) For example, this includes obtaining antibody-secreting immune cells (lymphocytes) from the spleen of a mouse. Furthermore, myeloma cells or phenotypes that have the ability to infinitely replicate antibody-secreting lymphocytes in cell culture. By fusing with converted cells, an immortal immunoglobulin-secreting cell line is created. The resulting fusion cells or hybridomas are cultured, and the resulting colonies are converted into the desired monoclonal cells. Screen for antibody production. Colonies that produce such antibodies are identified as clonies. The cells are then grown either in vivo or in vitro to produce a large amount of antibodies. The theoretical basis and practical methodology for fusing such cells are explained by Kohler and Milst. This is shown in ein, Nature 256:495 (1975).

[0115] Mammalian lymphocytes are affected by peptide antigens, such as KLH-CTEELRVRLASHLR. K-CONH2 (SEQ ID NO: 54) or KLH-CTEELRVSLASHLRK-CO It is at least 80%, 85%, 90%, or 95% identical to NH2 (SEQ ID NO: 55). Peptide antigens, which may optionally consist of one or multiple residues, up to a maximum of 20% of those residues. In vivo immunization of animals (e.g., mice) with peptide antigens containing numerical substitutions or deletions. Immunity can be achieved by the heparin-binding domain of APOE, for example. For example, TEELRVRLASHLRK (sequence number 3) or TEELRVSLASHLR At least 10 consecutive amino acids derived from a peptide containing K (SEQ ID NO: 2) and at least This may also include immunizing animals with peptides containing sequences that are 80% identical. Infection is repeated as needed, at intervals of up to several weeks, to obtain sufficient antibody titers. After the final antigen boost, the animals are slaughtered and spleen cells are extracted.

[0116] Mammalian myeloma cells or other fusion partners have the ability to replicate indefinitely in cell culture. The fusion is carried out by known techniques, for example, polyethylene glycol ("PEG") or other Using the fusion agent, the result is obtained (as incorporated herein by reference, Milstein (and see Kohler, Eur. J. Immunol. 6:511 (1976)). This immortal cell line (preferably, Although it is a weakling, it can also be derived from cells of other mammalian species, including rats and humans, without limitation. ) is the ability to grow rapidly, such as when an enzyme necessary for the utilization of a particular nutrient is deficient. They are selected to have good fusion ability and many such cell lines. This is known to those skilled in the art, and others are frequently described.

[0117] Procedures for producing polyclonal antibodies are also known. Typically, The antibodies are obtained from the blood of a New Zealand white rabbit, which is initially collected to obtain preimmune serum. It can be produced by administering the protein or polypeptide of the present invention subcutaneously to the gyi. The antigen is injected, for example, at six different sites in a total volume of 100 μl per site. Each injectable material contains a synthetic surfactant adjuvant pluronic polyol, or containing proteins or polypeptides after SDS-polyacrylamide gel electrophoresis It contains a finely ground acrylamide gel. Then, two weeks after the first injection, the rabbit Blood is drawn from the patient, and the same antigen is used to periodically boost the test three times at 6-week intervals. After that, each booster... Ten days after the test, a serum sample is collected. Then, polyclonal antibodies are captured. Affinity chromatography using the corresponding antigen to obtain the antigen is used to extract the antigen from serum. Collect. Finally, the rabbits were given, for example, pentobarbital 150 mg / kg IV. Euthanasia. This and other procedures for producing polyclonal antibodies are used by E. Harlo. This is disclosed in w, et. al., editors, Antibodies: A Laboratory Manual (1988).

[0118] The methods described herein involve chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, and two A stool that produces a hemispecific antibody, a fusion antibody, a labeled antibody, or an analogue of any one of these. It includes any one of the steps. The corresponding methods are known to those skilled in the art, for example, Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988 ) is described. When the derivative of the antibody is obtained by phage display technology. In addition, using a surface plasmon resonance method such as that used in the BIAcore system, this specification Phage antibodies that bind to the same epitope as any one of the antibodies described in the book. It can increase the efficiency of the body (Schier, Human Antibodies Hybridomas 7 (1996)). 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Chimeric antibody production is, for example, For example, it is described in International Publication No. 89 / 09622: Production of humanized antibodies. Methods for this include, for example, European Patent Application Publication No. 0239400 and International Publication No. Further details are provided in Specification No. 90 / 07861. The source of these antibodies is so-called heterologous antibodies. The production of heterologous antibodies such as human-like antibodies in mice The general principles for this are, for example, in International Publication Nos. 91 / 10741 and 94 / 02602. As described in specifications No. 96 / 34096 and No. 96 / 33735. As discussed, the antibodies described herein exist in various forms other than complete antibodies. For example, these include Fv, Fab, and F(ab)2, in addition to single-stranded molecules. For example, please refer to International Publication No. 88 / 09344.

[0119] Monoclonal antibodies are used in hybridoma, recombinant, and phage display technology. A wide range of types known in the art, including the use of rhology or combinations thereof. It can be prepared using the following technology. For example, monoclonal antibodies are available in this technology. It is known that, and for example, Harlow et al., Antibodies: A Laboratory Manual, Co. ld Spring Harbor Laboratory Press, 2nd ed. (1988);Hammerling et al., in: Monocl onal Antibodies and T-Cell Hybridomas Elsevier, NY, 563-681 (1981) The references are incorporated as a whole by reference, including those taught in the Hive It can be produced using redoma technology. When used herein, the term "monochrome" is used. "Ronal antibodies" are not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to a clone of any eukaryote, prokaryote, or phage. This refers to antibodies derived from a single clone containing the compound, and not to the method by which they are produced. Therefore, the term "monoclonal antibody" is produced through hybridoma technology. It is not limited to antibodies.

[0120] In known hybridoma methods (Kohler et al., Nature 256 (1975), 495), Relatively short-lived or lethal lymphocytes of mammalian origin, for example, as described herein. B cells derived from such mouse subjects fuse with immortal tumor cell lines (e.g., myeloma cell lines). They are combined, and therefore immortal, and capable of producing genetically encoded antibodies of B cells. It produces powerful hybrid cells or "hybridomas". The resulting hybrids Each individual strain is selected and diluted into a single-gene strain containing a specific gene for the formation of a single antibody. They are separated by regrowth and produce antibodies that are uniform against the desired antigen. And, in accordance with those pure genetic lineages, they are named "monoclonal."

[0121] The hybridoma cells prepared in this way are seeded onto unfused parental myeloma cells. Grow in a suitable culture medium containing one or more substances that inhibit the growth or survival of the organism. How many reagents, cell lines, and media for hybridoma formation, selection, and growth? It is commercially available from that source, and standardization protocols are well established for those skilled in the art. They are aware of this. Generally, the culture medium in which hybridoma cells are growing is used to target the desired antigen. The assay will be performed to assess the production of monoclonal antibodies produced by hybridoma cells. The binding specificity of the monoclonal antibody is determined by immunoprecipitation, as described herein. Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELSA), etc. The desired specificity, affinity, and / or activity are determined by an in vitro assay. After the identification of antibody-producing hybridoma cells, their clones were subjected to limiting dilution procedures. They can be subcloned and grown using standard methods; for example, Goding, Monoclonal Ant See ibodies: Principles and Practice, Academic Press, pp 59-103 (1986). Monoclonal antibodies secreted by clones can be found in culture medium, ascites fluid, or serum, for example, Protein A, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and It can be separated by standard purification procedures such as affinity chromatography.

[0122] In another embodiment, lymphocytes may be selected by microscopy and variable genes may be isolated. For example, peripheral blood mononuclear cells are isolated from immunized or innately immune mammals, such as humans. The culture can be cultured in vitro for approximately 7 days. The culture then meets the screening criteria. They are screened for specific immunoglobulins. Cells are isolated from the positive wells. Individual Ig-producing B cells are obtained by FACS or complement-mediated hemolytic plaque assay. They can be isolated by identifying them in (i). Ig-producing B cells are placed in a tube. The VH and VL genes can then be manipulated under a microscope and amplified, for example, using RT-PCR. The VH and VL genes were cloned into antibody expression vectors and expressed in cells. For example, it can be transfected into eukaryotic or prokaryotic cells.

[0123] Alternatively, antibody-producing cell lines are selected and cultured using techniques well known to those skilled in the art. Such techniques can be described in various experimental manuals and major publications. In this regard, the following technologies suitable for use in the present invention, including the appendix, are considered as a whole. The following is incorporated herein by reference: Current Protocols in Immunology, Colig an et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley It is described in and Sons, New York (1991).

[0124] Antibodies such as those mentioned above are synthetic genes that encode their listed amino acid sequences, for example. It can be produced by preparing and expressing the offspring. Any of the anti-ApoE antibodies Methods for producing such variants (e.g., including amino acid substitutions) are relevant in the art. These methods are well known. These methods involve an antibody or any part thereof (e.g., a flame Site-specific (and) prepared DNA molecules encoding the slash region, CDR, constant region Examples include oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis. These are some examples, but are not limited to them. Site-directed mutagenesis is well known in this field. (For example, Carter et al., Nucl. Acids Res., 13:4431-4443 (1985) and Ku See nkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987). PCR mutagenesis is also... Furthermore, it is suitable for creating amino acid sequence variants of starting polypeptides. Higuchi, i n PCR Protocols, pp.177-183 (Academic Press, 1990) and Vallette et al., Nucl. A See cids Res. 17:723-733 (1989). Another method for preparing sequence variants, cassette Mutagenesis is based on the technique described by Wells et al., Gene, 34:315-323 (1985). It is.

[0125] Antibodies can be produced in bacterial or eukaryotic cells. Some antibodies, for example... Fab can be produced in bacterial cells, such as E. coli cells. Antibodies can also be used with transformed cell lines (e.g., CHO, 293E, COS, HeLa), etc. It can be produced in eukaryotic cells. In addition, the antibody (scFv) is found in the Pichia genus (Pich ia) (See, for example, Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanse Yeast cells of the genera Hansenula or Saccharomyces have odor It can be expressed in order to produce an antibody or antigen-binding fragment of interest. Polynucleotides encoding the body are constructed, introduced into an expression vector, and then appropriate It is expressed in host cells. Prepare a recombinant expression vector and transfect host cells. Select the transformants, culture the host cells, and recover the antibodies using a standard method. Subbiological techniques are used.

[0126] In the field where antibodies are supposed to be expressed in bacterial cells (for example, E. coli) In this case, the expression vector should have properties that enable the amplification of the vector in bacterial cells. It is. In addition, JM109, DH5α, HB101, or XL1-Blue Whenever Escherichia coli (E. coli) is used as a host, the vector is transmitted to E. coli (E. coli). A promoter that can enable efficient expression in, for example, the lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al., Science, 240 It must have a T7 promoter, or :1041-1043 (1988)). Examples of vectors include, for example, the M13 series vectors, the pUC series vectors, and pB R322, pBluescript, pCR-Script, pGEX-5X-1(Ph armacia), “QIAexpress system” (QIAGEN), pEG FP and pET (when this expression vector is used, the host is preferably T7 RN) Examples include BL21, which expresses A polymerase. The expression vector is antibody secretion It may contain a signal sequence for this purpose. Regarding production in the surrounding material of Escherichia coli (E. coli), pe The LB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) is for antibody secretion. It can be used as a signal sequence. For bacterial expression, calcium chloride method or electro Poration can be used to introduce expression vectors into bacterial cells.

[0127] Antibodies are expressed in animal cells such as CHO, COS, and NIH3T3 cells. In this case, the expression vector is necessary for the promotion of expression in these cells. For example, the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Ac ids Res., 18:5322 (1990)), or containing the CMV promoter. Immunoglobulin or In addition to the nucleic acid sequence encoding that domain, the recombinant expression vector in host cells The vector includes sequences that control replication (e.g., origin of replication) and selection marker genes. It may have additional sequences. The selection marker gene is in the host cell into which the vector has been introduced. To facilitate selection (e.g., U.S. Patent Nos. 4,399,216 and 4,634,665) (See also No. 5,179,017). For example, typically, a selection marker gene is Resistance to drugs such as G418, hygromycin, or methotrexate, The vector is introduced into host cells. Examples of vectors with selection markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP Thirteen can be listed.

[0128] In one embodiment, the antibody is produced in mammalian cells. Examples of mammalian host cells include Chinese hamster ovary cells (CHO cells) (for example) However, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, DH Used in conjunction with FR selection markers, Urlaub and Chasin (1980) Proc. Natl. Acad. Sci dhfr as described in USA 77:4216-4220 - CHO cells are an example, derived from human fetuses. Kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells Cells, lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, as well as trans Examples include cells derived from genic animals, such as transgenic mammals.

[0129] The antibodies described herein are isolated from inside or outside host cells (e.g., culture medium) and are substantially pure. It can be purified as a homogeneous antibody. The isolation and purification methods commonly used in antibody purification are... The method for production may be used for the isolation and purification of antibodies, and is not limited to any particular method. No. Antibodies can be detected by methods such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, Solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis By appropriately selecting and combining dialysis and recrystallization, the material is isolated and purified. To obtain. Chromatography includes, for example, affinity chromatography and ion exchange. Chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization) zation: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Har (bor Laboratory Press, 1996) is one example. Chromatography includes HPLC and F This can be performed using liquid-phase chromatography such as PLC. Examples of columns used in matrixing include Protein A and Protein G columns. Examples of columns that use Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences) ) are examples. The disclosure also provides highly purified antibodies using these purification methods. include.

[0130] Antibody characterization The ApoE binding properties of the antibodies described herein may be determined by any standard method, for example, the following method. It can be measured by one or more of the following: OCTET®, surface plasmon resonance ( SPR), BIACORE® analysis, enzyme-linked immunosorbent assay (ELISA), E IA (enzyme immunoassay), RIA (radioimmune assay), and fluorescence resonance energy Ghee transfer (FRET).

[0131] Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raet her (1988) Surface Plasmons Springer Verlag;Sjolander and Urbaniczky (1991) Ana l. Chem. 63:2338-2345;Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705, and BIAcore International AB (Uppsala, Swed Information from SPR is provided in online resources. The equilibrium dissociation constant (K) for the binding of a body molecule to its target. d ), and K on and K of f It can be used to provide accurate and quantitative measurements of kinetic parameters, including [specific parameters]. ru.

[0132] Epitopes are also created using BIACORE chromatography technology, for different antibodies. In binding between wild-type ApoE and mutant ApoE (e.g., ApoEch), they By evaluating the competing capabilities, it can be directly mapped (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); Johne et See also al. (1993) J. Immunol. Methods, 160:191-198).

[0133] When using an enzyme immunoassay, the sample containing the antibody, for example, on the culture of antibody-producing cells, A clean or purified antibody is added to the antigen-coated plate. Alkaline phosphatase Secondary antibodies labeled with enzymes such as tase are added, the plate is incubated, and then washed. After purification, enzyme substrates such as p-nitrophenyl phosphate were added to evaluate antigen-binding activity. Absorbance is measured for this purpose.

[0134] Additional general guidance for evaluating antibodies, e.g., Western blotting and Immunoprecipitation assays are described in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Col. This can be found in Spring Harbor Press (1988).

[0135] Mutant ApoE proteins, peptides, and their fusion proteins This disclosure shows that, compared to the wild-type ApoE protein, 1 in the HSPG-binding domain A mutant ApoE protein or fragment containing amino acid substitutions at one or more positions. It provides. In some embodiments, the mutant ApoE protein or a fragment thereof , containing an amino acid other than arginine at position 136. In some embodiments, The mutant ApoE protein or its fragments contain serine, histidine, at position 136. or contains cysteine. Nuclei encoding the mutant ApoE protein or a fragment thereof. Acid sequences (e.g., DNA or RNA) and vectors containing such nucleic acid sequences are also Provided are mutant ApoE proteins or fragments thereof, such proteins or fragments. A nucleic acid encoding a fragment, and a vector containing the nucleic acid sequence, are used in dementia or mild cognitive impairment. Disorders associated with mental illness (MCI) (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia) (Solid dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease), neurodegeneration Useful for treating or preventing diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. That is the case.

[0136] In some embodiments, the mutant ApoE protein is the ApoEch protein (For example, ApoE2ch, ApoE3ch, or ApoE4ch proteins) A fragment of the ApoEch protein containing amino acid position 136 is also intended herein. An exemplary sequence of the full-length ApoE3ch protein is shown below. Arginine or Mutations to serine are shown in bold and double-underlined.

[0137] [ka]

[0138] In some embodiments, the methods disclosed herein involve mutant ApoE protein This allows the protein or fragments to cross the blood-brain barrier. (Variant ApoE protein) Alternatively, the fragments may be delivered using a nanocarrier, which contains polymer nanoparticles. Lipid-based nanoparticles, liposomes, micelles, dendrimers, and expressing the aforementioned proteins Examples include, but are not limited to, human cells and nanotubes (Dominguez et al. (See l. J Nanosci nanotechnol. 14(1):766-79, 2014). In some embodiments, The mutant ApoE protein or its fragments are delivered into the nasal cavity, carotid artery, or transmucosal delivery. (For example, intracarotid injection of hypertonic solution (arabinose or mannitol); Sanchez-Covarr ubias et al., Curr Pharm Des. 20(10):1422-49, 2014 and Miyake et al., World JO (See torhinolaryngol Head Neck Surg. 1(1):11-16, 2015) or chlorotoxin By using (see McCall et al., Tissue Barriers 2(4):e944449, 2014), delivery Hypothermia technology, receptor-mediated transport, cell-permeable peptides, and cell-mediated delivery are also being developed. Furthermore, it can be used to facilitate the crossing of the blood-brain barrier by the ApoE3ch protein. It is possible (see Pandey et al., Tissue Barriers 4(1): e1129476, 2016). For example, immunology Cells and stem cells (e.g., neural stem cells, induced pluripotent cells, and mesenchymal stem cells) are used in the healing process. It can be used to transport medical payloads across the blood-brain barrier. Nanoparticles are loaded. Mesenchymal stem cells can be used for this purpose (e.g., Roger et al. Bioma). See Terials 31:8393-401, 2010). Genetically modified stem cells (e.g., genetically modified mesenchymal stem cells). Stem cells can also be used (for example, Ebrahimi and Lalvand Hygeia.JDMed.vo See l.5 (1): 90-104, 2013). Chemical drug delivery systems (CDDS), e.g., He et a The method described in l., Cells, 7(4):24, 2018 can also be used. Additional methods for transporting proteins by interruption are known in the art.

[0139] Mutant ApoE proteins (e.g., mutant ApoE proteins described herein) Either of the following, for example, ApoEch) or nucleic acids that encode a fragment of it (for example, DNA) In some embodiments, ApoEch (or mRNA) is intended herein. Protein-coding mRNA can be modified to increase its stability (for example, This was described in Zangi et al., Nat Biotechnol. 31(10):898-907, 2013, and published by Moderna, Inc. Developed by; and described in Alberer et al., Lancet 390(10101):1511-1520, 2017. (Published and developed by Curevac and BioNTech).

[0140] Viruses containing DNA sequences encoding the mutant ApoE protein or fragments thereof. The vector intended herein is an exemplary vector encoding a full-length ApoE3ch protein. The cDNA sequence (including the signal peptide region) is shown below. From cytosine to Mutations to denine are shown in bold and double-underlined.

[0141] [ka]

[0142] Suitable vectors are known in the art. In some embodiments, Illus vectors are AAV vectors (e.g., Rosenberg et al., Hum Gene Ther (As described in Clin Dev 29(1):24-47, 2018). At the R136 position other than R136S The cDNA sequences encoding the ApoE protein containing the mutations are also included. In that embodiment, the mutation is R136H or R136C.

[0143] Peptides and fusion proteins In some embodiments, wild-type or mutant ApoE (e.g., as described herein) Contains the HSPG / heparin-binding domain of any of the mutant ApoE proteins. Peptides consisting of or are provided herein. In some cases, provided herein The amino acid sequence of the peptide is STEELRVRLASHLRKLRKRLLRDA DDLQK (Sequence ID 57), STEELRVSLASHLRKLRKRLLRDADD LQK (Sequence ID 58), RLVQYRGEVQAMLGQSTEELRVRLASHL RKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLA Includes or consists of sequences selected from the group consisting of SHLRKL (Sequence ID 60). These sequences and at least 80%, 85%, 90%, 95%, 96%, 97%, 98% Variants with 99% or more sequence identity can also be used. A fusion protein containing the peptide provided above is also disclosed. Several embodiments In this case, the fusion protein is a human antibody (e.g., human IgG1, IgG2, IgG3, It further includes the Fc region of IgG4. In some cases, the fusion protein is wild-type or The Fc region of a human antibody was added to the C-terminus of the HSPG / heparin-binding domain of the mutant ApoE. Includes. In some cases, the fusion protein is HSPG / HEPA of wild-type or mutant ApoE. The phosphorus-binding domain contains the Fc region of a human antibody at its N-terminus.

[0144] In some cases, the peptides and fusion proteins provided herein are HSPG / H It competes with the wild-type ApoE protein for binding to valine. In some cases, as specified herein. The peptides and fusion proteins provided are wild-type ApoE protein and HSPG / To reduce or regulate the binding between heparin molecules. In certain embodiments, this specification The peptides and fusion proteins provided are HSPG / of the wild-type ApoE protein. Inhibits and / or reduces heparin binding, and has one or more of the following properties: When administered to human patients or the following animal models, it reduces the severity of symptoms: Disorders associated with cognitive impairment or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular disorders) Dementia palsy, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease ), neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. Provided herein These characteristics of the peptides and fusion proteins are not known in the art. It can be measured in accordance with the law.

[0145] Anti-ApoE vaccines can be used to induce a protective immune response against ApoE. The following is also provided herein. In some embodiments, the anti-ApoE vaccine is One or more of the ApoE proteins provided herein, for example, and pharmaceutically acceptable Contains pharmaceutically acceptable adjuvants. Pharmaceutically acceptable adjuvants are known in the art. It is being done.

[0146] Pharmaceutical composition and method of administration The methods described herein use the antibodies and peptides described herein as active ingredients. This includes the use of a pharmaceutical composition containing either a fusion protein or a fusion protein.

[0147] Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. In short, the technical term "pharmaceutically acceptable carrier" refers to a carrier suitable for pharmaceutical administration, such as saline solution, solvent, or molten metal. Dispersing agents, coating agents, antibacterial and antifungal agents, isotonic agents and absorption retarders, and so This includes other items of the same type.

[0148] Pharmaceutical compositions are typically formulated to suit their intended route of administration. Examples of administration routes include parenteral, for example, intracranial, intranasal, carotid, intravenous, intradermal, and dermal. These include oral administration (e.g., inhalation) and transmucosal administration.

[0149] Methods for formulating appropriate pharmaceutical compositions are known in the art, for example, Re mington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and t he Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) Refer to the books in the series. For example, solutions used for parenteral, intradermal, or subcutaneous application. Alternatively, the suspension may contain the following components: water for injection, saline solution, fixative oil, polyethylene glycol Sterile diluents such as glycerin, propylene glycol, or other synthetic solvents; Antibacterial agents such as diethanolamine alcohol or methylparaben; ascorbic acid or sulfurous acid Antioxidants such as sodium phosphate; chelating agents such as ethylenediaminetetraacetic acid; acetates, cucumber A buffer such as sodium chloride or phosphate, and sodium chloride or dextrose A substance used to adjust the tonicity, such as hydrochloric acid or sodium hydroxide. It can be prepared with a base. Oral preparations are made from glass or plastic. It can be packaged in a sample, disposable syringe, or multi-dose vial.

[0150] Pharmaceutical compositions suitable for injection are sterile aqueous solutions (if water-soluble) or dispersions, and without May contain sterile powder for immediate preparation of bacterial injection solutions or dispersions. For intravenous administration. Suitable carriers include physiological saline, bacteriostatic water, and Cremophor EL (trademark) (BASF). Examples include Parsippany, NJ, or phosphate-buffered saline (PBS). In this case, the composition must be sterile and have easy needle penetration (syringabilitation). It should be fluid to the extent that y) exists. It should be stable under manufacturing and storage conditions. It should be protected from contamination by microorganisms such as bacteria and fungi. The carrier is, for example, water, ethanol, polyol (e.g., glycerol, propylene Recall, and liquid polyethylene glycol, and other similar substances), and appropriate It may be a solvent or dispersion medium containing a mixture of those substances. Appropriate fluidity is, for example, The use of coating agents such as lecithin reduces the required particle size in the case of a dispersion. Microbial activity can be maintained by maintaining the environment and by using surfactants. Prevention of use involves various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenoxyethanol. Achieved by thimerosal, ascorbic acid, thimerosal, and other similar substances. This can be done. In many cases, isotonic agents, such as sugars, mannitol, sorbitol, etc. It is preferable that the composition contains ethanol and sodium chloride. Sustained absorption of the injectable composition. This involves substances that slow down absorption, such as aluminum monostearate and gelatin. This can be achieved by including it in the composition.

[0151] The sterile injection solution is an active solution in a suitable solvent containing one or a combination of the components listed above. The required amount of chemical compounds are incorporated, and if necessary, the mixture is then filtered and sterilized. It can be prepared. Generally, dispersions involve incorporating the active compound into a sterile medium. Prepared by, the sterile medium is derived from the basic dispersion medium and the essentials listed above. It contains other necessary components. In the case of sterile powder for the preparation of sterile injection solutions, the preparation The preferred methods are vacuum drying and freeze-drying, which involve sterilizing and filtering the material beforehand. This process yields a powder of the active ingredient derived from the solution, plus any additional desired ingredients.

[0152] Oral compositions generally contain an inert diluent or an edible carrier. Therefore, the active compound may be incorporated together with excipients to form tablets, lozenges, or capsules. For example, it is used in the form of gelatin capsules. Oral compositions are also used in mouthwashes. It can be prepared using a fluid carrier for use as a pharmaceutically compatible compound. The composition may include a drug and / or an adjuvant as part of the composition. Tablets, pills, capsules Lozenges, troches, and other similar products contain the following ingredients or compounds with similar properties. It may contain any of the following: crystalline cellulose, tragacanth gum, or gelatin. Which binders; excipients such as starch or lactose, alginic acid, Primogel , or a disintegrant such as corn starch; magnesium stearate or Stero Lubricants such as tes; lubricants such as colloidal silicon dioxide; sucrose or sacchar Sweeteners such as corn; or peppermint, methyl salicylate, or orange flavor. Flavorings such as these.

[0153] Regarding administration by inhalation, the compound is administered using an appropriate spray, such as a gas like carbon dioxide. The form of aerosol spray from a pressurized container, dispenser, or sprayer containing the substance. It can be delivered by means of [unspecified method]. Such a method is covered by U.S. Patent No. 6,468,798. Examples include those listed in the specification.

[0154] Systemic administration of therapeutic compounds as described herein can also be obtained via the mucous membrane. For mucosal administration, the formulation uses a suitable penetrating agent for the barrier to which the drug is to penetrate. Penetrating agents are generally known in this field, and these include, for example, surfactants. Examples include bile salts and fusidic acid derivatives. Transmucosal administration is by nasal spray or This can be achieved by using suppositories or injections.

[0155] In one embodiment, the therapeutic compound is delivered in an implantable tablet and a microencapsulated delivery system. Along with a carrier that protects the therapeutic compound from rapid elimination from the body, such as a sustained-release formulation containing [unclear]. Prepared are biodegradable, biocompatible polymers, such as ethylene vinyl acetate and polyacid anhydride. Using polyglycolic acid, collagen, polyoltoester, and polylactic acid Such formulations can be prepared using standard techniques, or, for example, Alza Corporation and Nova Pharmaceuticals, It can be commercially obtained from Inc. Liposome suspension (against cell antigens) (Includes liposomes that have noclonal antibodies and target selected cells) These can be used as pharmaceutically acceptable carriers. These are, for example, U.S. 4. Prepared by a method known to those skilled in the art, as described in Specification No. 522,811. It is possible.

[0156] The pharmaceutical composition, along with instructions for administration, is packaged in a container, pack, or dispenser. It can be included within the sur.

[0157] CRISPR / Cas9-mediated gene editing of APOE By editing the APOE gene using a genome editing system, dementia and / or or disorders associated with mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular cognitive impairment) Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease ), treating or preventing neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. Methods for doing so are included in this specification. Generally, methods are described in this specification. The therapeutically effective dose of the genome editing system is required for such treatment or if necessary This includes the step of administering the drug to a target that has been determined to be undergoing the procedure. (Term: Genome editing system) " refers to any system having RNA-induced DNA editing activity. Genome editing in this disclosure The system consists of at least two components adapted from naturally occurring CRISPR systems. Elements: Includes gRNA and RNA-induced nucleases. These two components are finely detailed. It associates with a specific nucleic acid sequence in the cell and modulates the DNA within or around that nucleic acid sequence, for example single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or base substitutions. By producing one or more of these, it forms a complex that has the ability to edit. For example, For a complete description of genome editing systems, see, for example, International Publication No. 2018 / 026976. See pamphlet number. In certain embodiments, this disclosure is related to CRISPR / Cas9 genotypes. AAV vectors that code the video editing system, and the malfunctions described herein. The use of such vectors to treat or prevent harm is provided.

[0158] RNA-induced nuclease / Cas9 Various RNA-induced nucleases can be used in this method, for example, in International Publication No. 2018 / 026 As described in pamphlet No. 976, it can be used. Several implementation forms In this context, the RNA-inducible nuclease used in this method and composition is yellow grape It is either a cocci (S. aureus) Cas9 or a pyogenic streptococcus (S. pyogenes) Cas9. The example Cas9 protein shown can be obtained from any species, including bacteria or archaea, without limitation. They may be separated or derived therefrom. In some embodiments of this disclosure, Cas9 is separated The column shows the two nuclear localization sequences (NLS) at the C-terminus and N-terminus of the Cas9 protein. (For example, PKKKRKV (SEQ ID NO: 61)), as well as minipolyadenylation signals ( Modified to include (or polyA sequence). An example NLS is SV40 large T antigen. NLS (PKKKRRV (SEQ ID NO: 62)) and nucleoplasmin NLS (KRPA ATKKAGQAKKKK (Sequence ID 63)). Other NLSs in this art This is known, for example, Cokol et al., EMBO Rep. 2000 Nov 15; 1(5):411-415; Freitas See and Cunha, Curr Genomics. 2009 Dec; 10(8): 550-557. Exemplary poly The adenylation signal is TAGCAATAAAGGATCGTTTATTTTCATTG GAAGCGTGTGTTGGTTTTTTTTGATCAGGCGCG (Sequence No. 64) Yes. In some embodiments, RNA-induced nucleases are nuclease-inactive. This refers to Cas proteins (e.g., dCas9).

[0159] Guide RNA Target one or more sites in the HSPG-binding domain of wild-type ApoE. Guide RNAs (gRNAs) designed to do so are provided herein. In the application morphology, the gRNA undergoes a mutation at amino acid position 136, wild-type Apo It is designed to introduce a mutation in E. In some embodiments, as specified herein The provided guide RNA is wild-type APOE gene (e.g., APOE2, APOE3, Alternatively, it is designed to introduce the R136S mutation in APOE4, in which case, exemplify Typical guide RNAs can be found in Table 7. In some embodiments, double-strand breaks are repaired. A template for restoring and introducing the R136S mutation is also provided. An exemplary template sequence is: The following applies:

[0160] [ka] However, silent mutations that can cause the PAM motif to disappear are double-underlined, such as R136S. The codon corresponding to the mutation is shown in bold, and the PCR product is cleaved from the template-accepting clone. Silent mutations that can result in the SacI site are shown in italics.

[0161] In some embodiments, the guide RNA provided herein is wild-type APOE The target is exon 3 (amino acids 1-61) of a gene or variant present in the target. Designed to do so (therefore, the method involves the distribution of the APOE gene in the subject) Steps to determine the column, and using that array, target exon 3 in the target This may include the step of determining the appropriate guide RNA sequence for setting. In this embodiment, double-strand break repair via non-homologous end joining (NHEJ) is performed by ApoE This results in a short insertion or deletion that leads to knockout. An example for ApoE knockout. The exemplary guide RNA sequences are shown in Table 8.

[0162] Base editing In some embodiments, the APOE gene is modified using base editing techniques (e.g., Rees and L). iu, Nature Reviews Genetics 19, 770-788, 2018;Komor et al., Nature 533, 420-424 Edited using (as described in [reference]). In some embodiments, the guide RNA is , by using base editing, wild-type APOE genes (e.g., APOE2, APOE3, or A Designed to introduce the R136H mutation in POE4), in which case, an exemplary guide DRNAs can be found in Table 6. A base editor that converts C / G to A / T and A / T Adenine base editors that convert G / C can be used to introduce point mutations. Exemplary base editors include Komor et al., Nature 533, 420-424 and Gaudelli et al. Examples include those described in al., Nature 551, 464-471).

[0163] AAVdel system The method involves CRISP, which includes Cas9 nuclease and one or two guide RNAs. This includes delivery of the R / Cas9 genome editing system to the target area. Delivery methods include: For example, an adeno-associated ether containing, preferably, a Cas9 encoding sequence and guide RNA. One example is viral delivery using a virus (AAV) vector. Adeno-associated delivery is also possible. Viruses, due to their efficient replication and reproduction life cycle, act as helper viruses, unlike other viruses. Rus, for example, naturally occurring defects that require adenovirus or herpesvirus This is an example (see Muzyczka et al., Curr. Topics in Micro and Immunol. 158 for an overview). See 97-129 (1992). AAV vectors efficiently transduce various cell types, and in vitro Long-term expression of the introduced gene can be achieved with AAV vectors. It is widely used as a substitute in various animal models, as well as in clinical settings. And it has shown therapeutic effects; for example, Mingozzi and High, Nature Reviews Genetics 1 2, 341-355 (2011);Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-4 47; See Asokan et al., Mol Ther. 2012 April; 20(4): 699-708. At least 3 of AAV AAV vectors containing 00 base pairs can be packaged and recombinant Protein expression can be produced. For example, AAV2, AAV5, AAV2 / 5, AA V2 / 8 and AAV2 / 7 vectors are used to introduce DNA into photoreceptor cells. (For example, Pang et al., Vision Research 2008, 48(3):377-385; Khani et al.) , Invest Ophthalmol Vis Sci. 2007 Sep;48(9):3954-61;Allocca et al., J. Virol. 2 (See 007 81(20):11372-11380). In some embodiments, the AAV vector is international AAV capsid polypeptide described in application PCT / US2014 / 060163 It may include (or an array that codes for it); for example, International Application No. PCT / U Sequence IDs 1, 3, 5, 7, 9, 11, 13, 15, and S2014 / 060163 AAV capsid polypeptide having an amino acid sequence selected from a group of 17, The Cas9 sequence and the guide RNA sequence as described herein are included in the Illus particles. In some embodiments, the AAV capsid polypeptide is Anc80 Polypeptides, e.g., Anc80L27; Anc80L59; Anc80L60; An c80L62; Anc80L65; Anc80L33; Anc80L36; or Anc It is 80L44. In some embodiments, AAV is derived from the AAV2 serotype. It incorporates terminal inversion sequences (ITRs). Exemplary left and right ITRs are shown in International Publication No. This is shown on Table 6 of the 2018 / 026976 pamphlet. However, AAV 2 Numerous modified versions of the ITR are used in this field, and the ITR shown below is illustrative. It should be noted that this is not intended to be limiting. Modification of these sequences This is known in the art or is obvious to those skilled in the art, and therefore this It is included in the scope of disclosure.

[0164] Cas9 expression is driven by promoters known in the art. In that embodiment, expression is controlled by three promoters: cytomegalovirus (CMV), Elongation factor-1 (EFS), or human G protein-coupled receptor kinase-1 (hGRK1) It is driven by one of the following (which are specifically expressed in retinal photoreceptor cells). The nucleotide sequences for each of the promoters are from International Publication No. 2018 / 026 These are provided in Table 5 of pamphlet No. 976. Modifications of these sequences are for certain applications. Such modifications may be possible or desirable, and such modifications fall within the scope of this disclosure. .

[0165] gRNA expression in AAV vectors is controlled by promoters known in this field. Driven by polymerases such as the human U6 promoter in some embodiments. -ze III promoter. An exemplary U6 ​​promoter sequence is shown below:

[0166] [ka]

[0167] In some embodiments, the nucleic acid or AAV vector is the nucleic acid listed above. or one of the AAV vectors and at least 80%, 85%, 90%, 95%, 96%, 97% They share %, 98%, 99%, or more sequence identity.

[0168] The above AAV genome is packaged into an AAV capsid (e.g., AAV5 capsid). The capsid may be contained in a composition (e.g., a pharmaceutical composition) and / or against It can be administered to elephants. Exemplary pharmaceutically acceptable compositions comprising AAV capsid according to this disclosure are equilibrium A pharmaceutically acceptable carrier such as a saline solution (BSS) and one or more surfactants ( For example, Tween 20) and / or temperature-sensitive or inversely temperature-sensitive polymers ( For example, it may include pluronic. Other pharmaceutical formulations known in the art The element may also be suitable for use in the compositions described herein.

[0169] The compositions comprising AAV vectors as disclosed herein are not limited to those administered by injection (e.g., intracranial injection). The composition can be administered to the subject by any suitable means, including intranasal delivery. The concentration of the AAV vector within the syringe, in particular, the dead volume in the injection device, and the safe administration of the AAV vector. Considering the relatively limited volume that can be reached, a sufficient AAV dose can be delivered to the target brain. It is selected to ensure that it is administered. An appropriate dose is, for example, 1 × 10 11 Viral genome (vg) / mL, 2×10 11 Viral genome (vg) / mL, 3×10 11 Viral genome (vg) / mL, 4×10 11 Viral genome (vg) / mL, 5× 10 11 Viral genome (vg) / mL, 6×10 11 Viral genome (vg) / mL, 7×10 11 Viral genome (vg) / mL, 8×10 11 Viral genome (vg) / m L, 9×10 11 Viral genome (vg) / mL, 1×10 12 vg / mL, 2×10 12 Viral genome (vg) / mL, 3×10 12 Viral genome (vg) / mL, 4× 10 12 Viral genome (vg) / mL, 5×10 12 Viral genome (vg) / mL, 6×10 12 Viral genome (vg) / mL, 7×10 12 Viral genome (vg) / m L, 8×10 12 Viral genome (vg) / mL, 9×10 12 Viral genome (vg) / mL, 1×10 13 vg / mL, 2×10 13 Viral genome (vg) / mL, 3× 10 13 Viral genome (vg) / mL, 4×10 13 Viral genome (vg) / mL, 5×10 13 Viral genome (vg) / mL, 6×10 13 Viral genome (vg) / m L, 7×10 13 Viral genome (vg) / mL, 8×10 13 Viral genome (vg) / mL, or 9×10 13Viral genome (vg) / mL may be mentioned. Any appropriate A composition of a certain volume can be delivered to the subretinal or cochlear space. In some cases, the volume is retinal Selected to form blebs in the submembrane space, for example, 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters 10, 200 microliters, 250 microliters, 300 microliters That's right.

[0170] Explants are used to study gRNA and / or Cas9 expression after viral transduction. This is particularly useful for studying genome editing over relatively short intervals. These models also enable higher throughput, which may be possible in animal models. It can predict expression and genome editing in animal models and subjects. Large animal models (e.g., rabbits, pigs, non-human primates) are used to test drugs. For physical and / or toxicological studies, and to approximate volumes used in clinical practice. Under conditions and in volume, the systems, nucleotides, vectors, and compositions of this disclosure It can be used to test human anatomy and / or physiology. Because they are selected to reproduce relevant aspects, the data obtained in these systems are generally ( (Not necessarily) AAV vectors and combinations as disclosed in this disclosure in human and animal subjects Predict the behavior of mature plants.

[0171] Screening method (test compound) Disorders associated with dementia or mild cognitive impairment (e.g., Alzheimer's disease, vascular cognition) Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease ), treatment or prevention of neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. In order to identify useful active substances in this field, test compounds, such as polypeptides and polynes, are used. For screening creotides, inorganic or organic large or small molecule test compounds. Methods are included herein. In some embodiments, the test compound is ApoE This modulates the HSPG / heparin binding properties of proteins (e.g., wild-type ApoE protein). In some embodiments, the test compound is the ApoE protein (e.g., wild type). It reduces the HSPG / heparin binding property of the ApoE protein.

[0172] As used herein, "small molecule" refers to a molecule with a molecular weight of less than approximately 3,000 daltons. This refers to organic or inorganic molecules. Generally, small molecules useful in this invention are 3,000 dalts. It has a molecular weight of less than n (Da). Small molecules are, for example, at least about 100 Da to about 3 Up to ,000Da (for example, approximately 100 to approximately 3,000Da, approximately 100 to approximately 2,500Da, Approximately 100 to 2,000 Da, approximately 100 to 1,750 Da, approximately 100 to 1,500 D a, about 100 to about 1,250Da, about 100 to about 1,000Da, about 100 to about 750D a, approximately 100-500Da, approximately 200-1500, approximately 500-1000, approximately 300 It can be around 1000 Da, or between 100 and 250 Da.

[0173] The test compounds are, for example, natural products or combinatorial chemistry libraries. It can be a group. A set of diverse molecules can have different characteristics such as charge, aromaticity, hydrogen bonding, mobility, and size. It is used to cover a variety of functions such as length, side chain length, hydrophobicity, and rigidity. It is. Combinatorial techniques suitable for synthesizing small molecules include, for example, Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molec Examples can be found in *Urular-Weight Compound Libraries*, Pergamon-Elsevier Science Limited (1998). As is known in the art, "split and pool" or This includes technologies such as "parallel" synthesis techniques, solid-phase and liquid-phase techniques, and encoding techniques. (For example, see Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, several That small molecule library is commercially available. Several suitable small molecule test compounds are whole and The following is incorporated herein by reference in U.S. Patent No. 6,503,713 It is listed.

[0174] The library screened using the method of the present invention contains various types of test compounds. It is possible. A given library is a set of structurally related or unrelated experiments. It may contain a compound. In some embodiments, the test compound is a peptide or a peptide It is a mimetic molecule. In some embodiments, the test compound is nucleic acid.

[0175] In some embodiments, the test compound and its library are the first test compound For example, the first one is structurally similar to a known natural binding partner of the target polypeptide. The structure of the test compound, or the first small compound identified as having the ability to bind to the target polypeptide. The molecule is used, for example, by methods known in the art or described herein. This involves systematically altering the structure and correlating the resulting biological activity with its structure, for example, structure- This can be obtained through activity relationship studies. As those skilled in the art will understand, such a structure - There are various standard methods for establishing activity relationships. Therefore, in some cases, the research is extensive. In some cases, this may be empirical, and in other cases, the three-dimensional structure of an endogenous polypeptide or a part thereof. However, it can be used as a starting point for the rational design of small molecule compounds. For example, In the application method, a general library of small molecules can be used, for example, by the method described herein. It is used for screening.

[0176] In some embodiments, the test compound is used in a test sample, for example, one or more A The sample containing poE protein was applied, and the efficacy of one or more test compounds was determined. The effect (e.g., the HSPG / heparin binding affinity of the ApoE protein) is evaluated. The ability of test compounds to modify the HSPG / heparin binding affinity of oE proteins is, for example, , heparin Sepharose column, or HS of ApoE as described herein This can be evaluated using antibodies that specifically recognize the PG-binding domain. In the embodiment described herein, test compounds such as those described herein are screened. The method involves one or more HSPG binding sites of wild-type or mutant ApoE or This refers to binding to one or more sites of allosteric regulation of HSPG binding as described herein. Tests that modify (e.g., inhibit or reduce) antibody binding as described above. This includes evaluating the capabilities of the compound. In some embodiments, the test compound is Apo It competes with antibodies described herein for E-binding.

[0177] In some embodiments, the test specimen is a fault as described herein. An Nvibo model, or a model derived from it (for example, a sample taken from it). In that case, animal models, such as rodents like rats, can be used.

[0178] Screened by the methods described herein, ApoE and HSPG / heparin Test compounds that have been determined to reduce or modify the bonding are considered candidate compounds. This can be done. For example, disabilities, for example, dementia and / or mild cognitive impairment (for example, Luzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease (Huntington's disease or related conditions), neurodegenerative diseases, cerebrovascular diseases, brain injuries, In an in vivo model of retinal degeneration or retinal damage, screening is performed for the said impairment. For example, it has been determined that it produces a desirable effect on one or more symptoms of the aforementioned disorder. Candidate compounds can be considered as candidate therapeutic agents. Candidate therapeutic agents are once set up in a clinical setting. If screened, it is a therapeutic agent. Candidate compound, candidate therapeutic agent, and treatment The agent may be optionally optimized and / or derivatized and manufactured with physiologically acceptable excipients. It can be formulated to form a pharmaceutical composition.

[0179] Therefore, the test compound identified as a "hit" in the first screening (for example) Select a test compound that produces a desirable effect on the disorder, and evaluate its binding affinity, binding activity, and specificity. or systematically change, for example using rational design, to optimize other parameters. Such optimization can also be performed using the methods described herein. It can be leaned. Therefore, in one embodiment, the present invention is in the art A first library of compounds is obtained using known and / or methods described herein. The screening step involves identifying one or more hits in the library. The step involves subjecting those hits to a systematic structural change, and structurally related to the hits. The steps of creating a second library of compounds, and the present invention This includes a step of screening using the method described in the details.

[0180] The test compounds identified as hits are those described herein for dementia and / Alternatively, disorders associated with mild cognitive impairment, such as Alzheimer's disease, vascular dementia, and Lewy body dementia. The onset of small body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease is also possible. Or useful in treating, preventing, or slowing the progression of neurodegenerative diseases, brain To treat and prevent the onset or progression of vascular disease, brain injury, retinal degeneration, or retinal damage. These can be considered candidate therapeutic compounds that are useful in delaying or mitigating the disease. "Hit" Various techniques useful for determining the structure, such as NMR, mass spectrometry, and electron capture detectors. Gas chromatography, fluorescence, and absorption spectroscopy, equipped with the methods described herein, are described herein. It can be used for this purpose. Therefore, the present invention also allows for the following by the method described herein: Compounds identified as "HITs," as well as methods for administering them, and as described herein. This includes use in treating, preventing, or delaying the onset or progression of the listed disorders.

[0181] The test compounds identified as candidate therapeutic compounds are for the treatment of disorders as described herein. Further screening can be performed by administering the drug to animal models of disorders related to either of these conditions. Animals can observe changes in the disorder, such as parameters of the disorder, or clinical outcomes. Improvements in the specified parameters can be monitored.

[0182] Treatment method The methods described herein are for the treatment of dementia and / or mild cognitive impairment. Treatment of the onset or progression of neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. Methods for inducing, preventing, or delaying dementia and / or impairments associated with mild cognitive impairment include Alzheimer's disease, vascular dementia, and Lewy body dementia. It is dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease. Additional non-limiting examples of sexually transmitted diseases include prion diseases, motor neuron diseases, and amyotrophic lateral sclerosis. ALS is one example. Non-specific examples of cerebrovascular diseases include stroke, carotid artery stenosis, Examples include vertebral artery stenosis and aneurysms. Non-limiting examples of brain injury include traumatic brain injury and These include acquired brain injury. Retinal degeneration such as glaucoma and age-related macular degeneration is caused by amyloid-beta It may also include neurofibrillary tangle toxicity, retinal degeneration and neurodegeneration (e.g., Alzheimer's disease) The relationship between them has been established (for example, McKinnon, Frontiers in Bioscience 8, s1140-115). 6, 2003; Johnson et al. PNAS 99(18) 11830-11835, 2002; and Sivak, Investigativ (See e Ophthalmology & Visual Science, 54(1) 871-880, 2013). Therefore, neurodegeneration Treatment for this condition can be used to treat retinal or optic nerve degeneration.

[0183] The method involves antibodies, peptides, fusion proteins, or gels as described herein. Nom editing system, a therapeutically effective amount, for those requiring such treatment, This includes administering the drug to subjects who have been determined to be in need of it.

[0184] The methods described herein carry a risk of causing any of the disorders described herein. It is also useful for subjects with APOE. Subjects at risk of developing Alzheimer's disease should be treated with APOE. Homozygous or heterozygous for 4 alleles, autosomal dominant Alzheimer's disease Mutations that cause this (e.g., amyloid-beta precursor (APP) gene, PSEN1 gene, (or mutations in the PSEN2 gene), trisomy 21 (for example, cognitive impairment is developmentally This may include holders of the subject (which is only a subject). Subjects at risk of developing Alzheimer's disease are, Those who have a polygenic risk score associated with an increased risk of developing the disease, and Brain imaging or other biomarkers of Alzheimer's disease (e.g., biomarkers in body fluids) This may also include those with evidence of the manufacturer. This specification aims to prevent the occurrence of the disorders described herein. The method to delay it is for those who are not at risk of developing the above-mentioned disorders, for example, those over 50 years old. (For example, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, It may also be useful for any subject (over 95 years old).

[0185] When used in this context, "to treat" means as described herein. This means achieving remission of at least one symptom of a disorder associated with the disorder. Alzheimer's disease is characterized by fibrosis, amyloid aggregation, and cognitive decline; Therefore, the treatment involves reducing fibrosis and / or amyloid aggregation in the brain, and increasing tau concentration. Reduced formation of cysts, improved brain metabolism, and improved cognitive neuroscientific function and / or cognitive abilities. It can happen. [Examples]

[0186] The following embodiments are provided to better illustrate the claimed invention and to extend the scope of the invention. It should not be interpreted as limiting. To the extent that specific materials are mentioned, it is simply This is for illustrative purposes only and is not intended to limit the present invention. Those skilled in the art will know the inventive ability. Without exhibiting the same effect and without departing from the scope of the present invention, equivalent means or reactants It is possible to develop this.

[0187] [Example 1] APOE3 Christchurch R136S mutation in PSEN1 mutation carriers Identification of differences material and method: Clinical evaluation: University of Antioquia, Massachusetts tts General Hospital and Schepens Eye Res search Institute of Massachusetts Eye and Ear's institutional ethics committee approved this study. As with all study participants, the proband The patient provided written informed consent. Clinical assessment methods and neuropsychology The appropriate tests were performed as described in Table 1. PSEN1 E280A genotype identification. As previously stated 1 ,went.

[0188] All clinical measurements are conducted by the University of Antioquia (Medell). The assessment was undertaken in Colombia by trained physicians and psychologists. It was conducted in Spanish. The neurocognitive tests included a comprehensive multi-domain assessment. Part of the test is the Mini Mental State Examination, which has been adapted for this Colombian population. -Mental State Examination (MMSE), Clinical Dementia Assessment (CDR), and Spanish bar consortium to establish registration for Luzheimer's disease battery It was John 2 Additional tests were performed within 6 months of the brain imaging, at Yesav. Yesavage Geriatric Depression Scale 3 and functional evaluation progress Functional Assessment Staging Test 4 It was made from.

[0189] A detailed ophthalmological evaluation was conducted, including visual acuity assessment, slit-lamp and indirect optometry. Optos Panoramic 200Tx Imaging System (Optos P Ultra-wide-angle fundus and eye using LC (Dunfermline, Scotland, UK) A bottom autofluorescence image was obtained. In addition, Spectralis SD-OCT (Hei delberg Engineering, Heidelberg, Germany) Cirrus HD-OCT with AngioPlex (Carl Zeis OCT angiography was also performed at Meditec, Dublin, CA.

[0190] The PSEN1 E280A mutation carrier has two copies of the APOE3ch variant. After this was discovered, further research was conducted. Hyperlipoproteinemia type III (relatively A D: 5-10% of homozygous individuals have the protective APOE2 allele, and not all of them. This condition is found in most APOE3ch holders. 5 To explore the possibility of ) on an empty stomach A serum lipid panel was performed.

[0191] Finally, clinical and neurological information from the AD Genetics Consortium. Using an analysis of data from pathologically validated AD cases and controls, APOE2 Is homozygosity for Rel associated with an exceptionally low risk of late-onset AD dementia? The matter was revealed.

[0192] Whole exome sequencing: Whole exome capture and sequencing for variant discovery. The quenching was performed using Illumina chemistry; previously associated with AD. Rare variants in the identified genes, occurring at a frequency of less than 1%, were considered candidate stags in this study. The gene exon region and splice site were investigated. Specifically, gene modification factors were examined. Rare DNA variants within a link (5 bp in an intron) (minor allele frequency) <1% were identified using bioinformatics tools. Whole exome live We constructed a larium and paired 151bp pairs using an Illumina HiSeq 4000 sequencer. Sequencing was performed using end reads. The library construction was modified as follows: The previously described protocol 6 The procedure was performed using [a specific method]. A genomic DNA input of 10 μg was used. The dose was reduced from 3 μg to 50 ng in solution L and enzymatically sheared. It was double-indexed. Illumina paired end adapter, with 8 bases embedded within the adapter Replaced with a fork-shaped adapter of Palindrome having a DEX array, adapter chain Hybrid selection in solution was applied to each end for bonding. 38Mb target territory (29 This was performed using the Illumina Rapid Capture Exome Enrichment Kit with Mb Bait. The target region is interval 98 in the Refseq exome database. It included 3%. Dual-indexed libraries up to 96 before hybridization. The samples were pooled into groups of up to 1. The concentrated library pool was then subjected to streptavidin bile ducts. After elution from the cluster, the levels were quantified using PicoGreen and then standardized. - For amplification and sequencing, a fork-type indexing adapter is used. The prepared library was analyzed using quantitative PCR (KAPA biosystems). Quantitated and processed using a Hamilton Starlet liquid handling system, 2 nM Standardized for this, using the Hamilton Starlet liquid handling system Then, the same volume was pooled. After that, the pool was denatured in 0.1N NaOH. The sample was then stripped using the Hamilton Starlet liquid handling system. The solution was diluted into a tube. Cluster amplification of the template was performed using an Illumina cBot. The procedure was carried out according to the manufacturer's protocol (Illumina). The flow cell was Hi Sequencing with the Seq 4000 Sequencing-by-Synthesis Kit The data was singled and then analyzed using RTA2.7.3.

[0193] Exome sequencing data, Center for Personalize d Medicine(CPM)Clinical Genomics Laborat ory and Translational Genomics Research In Bioinformatics of Clinical Exome Sequencing at the Institute Center Processed and analyzed in a pipeline. In short, Edi with default parameters Using co Genome's Dragen genome pipeline, sequence alignment We implemented variant calling using open-source software SAM. tools and bcftools (samtools.github.io / ) Used with a custom script in the set, ExAC (Exome Aggregation) on Consortium, exac.broadinstitute.org / ) Based on the rel frequency, coverage is determined and the initial variant filtering is performed. The group was 7 Sequence alignment was performed on the human hs37d5 decoy genome. 8 To identify potential modifying factor variants, we performed a major gene sequencing of 15 genes. The strike, two HPO terms: HP:0002511, Alzheimer's disease; HP:000 3584, created based on the late-onset type. These genes are AAGAB, ABCC8, A KT2, APOE, APP, BEAN1, GATA1, GCK, HMGA1, HNF1B These were HNF4A, LDB3, PAX4, PSEN1, and PSEN2. 9 .these The exon region and splice site junction (5 bp into the intron) of the gene Further, rare DNA variants within (minor allele frequency <1%) can be identified using commercially available tools (Ca Annotation and analysis were performed using rtagenia v5.0). Sequence changes were analyzed using Hu man Genome Variation Society (HGVS v2.0) life The report was submitted in accordance with the nomenclature guidelines.

[0194] Whole-genome sequencing: Whole-genome sequencing (WGS) and Genomize R-analysis (v 10.1.0) is used to rule out other possible protective genetic factors. To help with this, include those associated with a lower risk of Alzheimer's disease. We conducted a comprehensive and unbiased ranking of other possible genetic risk modifiers. 10 Regarding processing WGS data, the same dragen pipeline as above is used. The data was aligned to the GRCh37 decoy genome (hs37d5). The variants called at a depth of <10x were filtered out, and then, Ensembl's Variant Effect Predictor (VEP) tool Annotation was performed using [tool name]. The VEP version used was v93. Afterwards, [tool name] The filtered and annotated set of ants is used for Genomizer analysis. Edited for this purpose.

[0195] APOE Structural Displays: NGL Vi from RCSB PDB (rcsb.org) Using ewer, PDB 2L7B, previously published structure 11 Obtain an image and modify it. did 12 .

[0196] APOE genotype identification by Sanger sequencing: Mixing of reactants for amplification treatment This was carried out in a 50 μL volume containing the following components. 13 :1×Pfu Ultra I I Hostart Master Mix, 1 μL of each primer (10 μmol / L) )(Forward Primer F: 5'-AGCCCTTCTCCCCGCCTCCCACT GT-3' (SEQ ID NO: 67) and reverse primer: 5'-CTCCGCCACCT GCTCCTTCACCTCG-3' (SEQ ID NO: 68), 5% DMSO, and 1 μ L genomic DNA (100 ng / μL). PCR cycling at 95°C for 2 minutes. Denature, followed by denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, and at 72°C The process involved 35 cycles of 40-second extension, followed by a final extension of 5 minutes at 72°C. PCR The substance was purified using the QIAquick gel extraction kit from Qiagen, and MGH C The CIB DNA core allows Applied Biosystems' 3730 The sequencing was performed using an XL sequencer.

[0197] MRI and PET imaging: Pittsburgh Compound B(P iB), Flortausipil (FTP) positron emission tomography (PET), and structural magnetic Air resonance imaging (MRI) measurements are performed at Massachusetts General Hospital. It was obtained via pital, as previously described. 14 Massachusetts General Hospital and Banner Alzheimer's I Analyzed at the National Institute. Fluorodeoxyglucose PET images were obtained at the National Institute. Acquired in the city of Antioquia, Colombia, previously described As 15 The imaging data from the cases was analyzed. From younger PSEN1 E280A mutation carriers who developed MCI at the expected age Imaging data, and imaging from mutant carriers whose cognitive abilities are not impaired. It was compared with Gdata.

[0198] The MRI was performed using a 3T Tim Trio (Siemens) and as previously described. As if 16-19 Identify the gray matter and pia mater surface, and define the region of interest (RO) as follows. I) Magnetization-prepared high-speed processing with Freesurfer (FS) to enable partitioning Gradient echo (MPRAGE) included: cerebellar gray matter, hippocampus, and the following Br AAK stage-related cortex: entorhinal, parahippocampal gyrus, inferior temporal, fusiform, posterior cingulate

[0199] In MGH, a radiochemical yield of 14±3% was obtained at the end of the synthesis (60 minutes later). 18F-Flortausipyr (FTP) has a specific activity of 216±60 GBq / μmol. ) was prepared and confirmed for human use (Shoup et al., 2013). 11C-Pitt Sburgh Compound B was prepared as previously described. 16 , P ET images were acquired. All PET images were obtained using Siemens / CTI (Knoxvil). le, TN)ECAT HR+ Scanner (3D Mode; 63 Image Planes; 15.2cm Body Axis) Directional field of view; acquired using 5.6 mm axial transverse resolution and 2.4 mm slice spacing. . 11C PiB PET involves an 8.5-15 mCi bolus injection, followed immediately by 69 Acquired using dynamic acquisition over 60 minutes per frame (12 x 15 seconds, 57 x 60 seconds). 18F FTP is measured in 4 x 5 minute frames with a 9.0-11.0 mCi bora Acquired 80-100 minutes after injection. PET images were reconstructed and attenuated. Each frame was evaluated to verify sufficient counting statistics and the absence of head movement.

[0200] 18F FTP-specific binding occurs at the FS ROI, with the FS cerebellar gray matter ROI as a reference. Using I, the previous report 19 Similarly, the standardized uptake ratio (SUVR) for the cerebellum is used. It was expressed as follows. For voxel analysis, the MPRAGE of each target is SPM8 (SPM) The template is registered in MR, and spatially transformed SU to account for individual anatomical differences. VR PET data was smoothed with an 8mm Gaussian kernel. 20 Confusing hippocampal signals To consider possible 18F FTP off-target binding in the choroid plexus, this The inventors, as previously reported 21 Linear regression is used to regress the choroid plexus. there was.

[0201] 11C PiB PET data uses cerebellar gray matter as a reference tissue for distribution volume ratio (D Represented as VR; Log applied to data from 40 to 60 minutes after injection. To calculate the local DVR for each ROI using a graphical method, A time-activity curve was used. 16,22 11C PiB retention is as previously described. like 23,24 The large skin includes the frontal, lateral temporal, and posterior corpus callosum cortex (FLR). The evaluation was performed using a qualitative ROI aggregate.

[0202] 18F-Fludeoxyglucose PET is 64-section PET / computed PET. In a layered imaging system (Biograph mCT; Siemens), Intravenous administration of 5 mCi (185 million Bq) of 18F-fludeoxyglucose was used. This was performed after a 30-minute rest period in a dark room to allow for radioactive tracer absorption, followed by... A 30-minute dynamic emission scan (six frames at 5-minute intervals) was then performed. The images were reconstructed using computed tomography attenuation correction. Precuneus glucose relative to the whole brain. The brain metabolic rate (CMRgl) ratio for each participant is calculated using 18F-fludeoxyglucose. Automated brain mapping algorithm (SP) from bilateral regions of interest (ROI) in PET images M8;fil.ion.ucl.ac.uk / spm / software / spm8) The following was used to characterize the data: The ratio of hippocampal volume to total intracranial volume was used for each participant's T1-weighted M2. From the bilateral ROI in the R image, FreeSurfer(surfer.nmr.mg The images were characterized using h.harvard.edu. All images were ROI-featured. It was visually inspected to verify it.

[0203] Amyloid aggregation studies: Humans with and without the Christchurch variant. ApoE3 protein fragment (including carboxyl-terminal domain and histidine tag) However, it is synthesized and purified in bacteria (Innovagen), and thioflavin T (Sen soLyte® ThT β-amyloid (1-42) aggregation kit, catalog number Using (model number AS-72214), the Aβ of these proteins was analyzed in vitro. 42 Different aspects of aggregation It was used to evaluate the effect. For this assay, 96 clear unbound samples were taken. In a well plate, 55 μM Aβ 42 10 μM wild-type ApoE3 or variant The variant 136Arg was added to a solution of either Ser ApoE3 protein. The sample was mixed with 2 mM thioflavin T dye, and the fluorescence was measured at Ex / Em = 440 / 484. The readings were taken intermittently over a two-hour period. The plate was kept at 37°C and read. The device was shaken for 15 seconds between taking and reading.

[0204] Full-length ApoE3 proteins with and without the Christchurch mutation are also included. In addition, Flp-In(TM) T-REx(TM) 293(Thermo Fisher S (Scientific) Expression in mammalian cells by transient transfection Furthermore, the effect of these proteins on Aβ42 aggregation was previously published in the Split Shift Confirmed using a gelase complementation assay. 26 The latter analysis involves WT, or site-specific mutations. As an APOE3 containing the Christchurch variant introduced by heterogeneity Addgene product (plasmid number 87086) 27 Using human APOE3 expression from ) The procedure was carried out. Reagents for the luciferase assay were purchased from Promega. .

[0205] result Approximately 1,200 Colombians carry the presenilin 1 (PSEN1) E280A mutation. And approximately 4,600 non-carriers were identified, and together they had autosomal dominant Alzheimer's. Constituting the world's largest known family with ADAD 28,29 Mutation carrier Those typically experience symptoms between the ages of 44 (95% CI, 43-45) and 49 (95% CI, 49-50) ) The onset of mild cognitive impairment (MCI) and dementia at the respective median ages. 30,31 Autosomal dominant AD (ADAD) variant, in which cognitive abilities remain intact into old age. Studying heterogenes can help in discovering risk-reducing gene variants. 32 .this Characterizing AD biomarkers in these individuals indicates that these genes are phases of their development. This can help provide information about potentially targetable mechanisms that exert a counter-defensive effect. The inventors have shown that MCI can be managed for approximately 30 years after the median age of onset, up to her 70s. We identified individuals who carried the PSEN1 E280A mutation but did not develop symptoms.

[0206] This study required participants to provide written information in accordance with the guidelines of the institutional review committee. It was done with her consent (her exact age and other identifying information were not disclosed). (Removed to protect anonymity and confidentiality). Participants were amyloid-beta. 42 (Aβ 42 ) It was confirmed that she has an overproduction of the PSEN1 E280A mutation, and her 70 Family informants have confirmed that cognitive abilities have not been impaired up to the age of 20, Furthermore, during the 24-month period of the annual evaluation, the criteria for determining MCI (Mild Cognitive Impairment) 33 She met the basic requirements. They remain completely independent in essential and instrumental activities of daily living, There were no obvious signs of deterioration in her ability to perform the action. At the time of the intake evaluation, Her memory impairment was limited to recent events, and her neurological examination was normal. The neuropsychological test scores, adjusted according to age and education, were re-evaluated during the 24-month assessment period. Raw memory, relatively stored recognition memory, initial learning ability, naming ability, visuospatial ability, and The patient exhibited a preferential impairment in written fluency skills, as well as relatively stable cognitive abilities (Table 1).

[0207] [Table 6]

[0208] Whole exome sequencing confirmed her PSEN1 E280A mutation, and she Additionally, there is the rare APOE3 Christchurch R136S (APOEch) mutation. It was discovered that it had two copies. Sanger sequencing confirmed the latter finding. Using whole-genome sequencing and Genomizer analysis, all potentially important Furthermore, rare and common variants were comprehensively identified and ranked. 34 Using this approach, the PSEN1 E280A mutation was identified as a major risk factor for the participant. It was confirmed to be a factor, and APOE3ch homozygosity was her main resistance factor. It was confirmed that this was the case.

[0209] APOE, the main susceptibility gene for late-onset AD, has three common alleles. It has APOE2, 3, and 4). Compared to the most common APOE3 / 3 genotype. Furthermore, APOE2 is associated with a lower risk of Alzheimer's disease and a higher age at the onset of dementia. It is attached 35 Each additional copy of APOE4 carries a higher risk and It is associated with a younger age at the time of onset. 36,37 AP, which corresponds to codon 154. OEch variant (substitution of arginine to serine at amino acid 136 (136A rg→Ser)) 38 This includes the two APOE3 alleles of this participant, as well as the general APO It can be present in any of the E alleles. 39 Figure 1 shows the structure of the wild-type ApoE3 protein. This indicates the Dell. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are It is emphasized. APOE4 (C112R), APOE3ch (R136S), and A The amino acid positions for the POE2(R158C) variant are shown.

[0210] The APOE3ch variant reports approximately 180,000 exomes. It was not present in the AD database or the ExAC database. R136S was previously HL It was identified in APOE2 individuals with P III, but its latency in the progression of AD is questionable. The present effect had not been reported previously. 40 The inventors have found a way to delay the age of onset (age of onset) DNA samples from two other PSEN1 E280A carriers (aged 62 and 70) The samples were sequenced using whole-genome sequencing. All of these individuals were APO Neither the E3 R136S variant nor the APOE2 was present in this lineage previously. It has been shown to delay the onset of the disease.41 .

[0211] A possible association between the APOE3 R136S mutation and delayed age of onset in Alzheimer's disease has been confirmed. To that end, the inventors all have been over 50 years of age and have been using APOE3 R136S Whole-genome sequencing was performed on four descendants of the proband patient who is expected to have neurological Academic and neuropsychological tests were performed. Figure 2 shows the results from control, proband, and offspring samples. The following shows typical Sanger sequencing results for APOE. Top row: C112 homozygous The column is shown for all cases. Middle row: R136S homozygous sequence is found in the control individual or This is shown in the left panel. The central panel shows homozygous individuals that produce the R136S mutation. The changes are shown. The right panel shows examples of R136S heterozygous mutations in the proband's offspring. The R158 homozygous sequence is shown in all cases. Figure 3 shows the family tree of the proband. The circle represents a woman, the square represents a man, and the rhombus represents a gender that is hidden for privacy reasons. The diagram represents individuals with MCI (Mild Cognitive Impairment), the arrowhead shape depicts the proband individual, and the shading indicates a history of dementia. This shows individuals that possess [the characteristic]. Deceased individuals are marked with crossed bars. Individual A The POE and PSEN1 genotypes are appropriately indicated to protect anonymity. Other unidentified The genetic or epigenetic factors of knowledge are related to the PSEN1 E in these two individuals. While it may have contributed to a later onset age of cognitive impairment in holders of the 280A, the inventors The APOE3 R136S variant mitigates the effects of amyloid-beta accumulation in the brain. Subsequently, it delays the onset of tau pathology, neurodegeneration (i.e., brain atrophy), and symptoms. This proposes modifying the AD phenotype.

[0212] Within the low-density lipoprotein receptor (LDLR) binding domain of APOEch and APOE Other carriers of rare mutations were generally observed in 5-10% of APOE2 homozygotes. Similarly, those with hyperlipoproteinemia type III (HLP-III) 43,44 .this The participants in the report had HLP-III and APOEch and triglycerides. This included elevated total cholesterol levels (see Table 2).

[0213] [Table 7]

[0214] Detailed laboratory precision testing was performed on the inventor's proband individual possessing APOE3 R136S. This also showed abnormal lipid profiles in three of the four descendants (Table 3). These four subjects had high levels of total cholesterol and triglycerides. High-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are two types of offspring. It was higher at 400 mg / dL (due to the accuracy of the indirect method of lipid profiling). In the proband and one of its offspring, triglyceride levels higher than the threshold were observed. It could not be measured (Table 3). Further analysis using direct enzyme testing was needed for these two individuals. The individual showed higher than normal LDL levels. One of its offspring had APOE3 R136S. Despite having APOE4, the HLP had a lipid profile within the normal range. Incomplete penetrance of III previously occurred in APOE2 mutation carriers and R136S mutation carriers. Let me report on this. 45 The inventors have identified diabetes, obesity, and alcohol in these subjects. Secondary causes of lipid disorders such as drug addiction, renal impairment, or thyroid disease were ruled out. None of the individuals with the condition had xanthomas, which are diagnostic indicators for HLP III, nor did they have cardiovascular disease. These subjects exhibited abnormal lipid profiles and APOE3 R136S mutations. This combination is consistent with the diagnosis of familial HLP III.

[0215] [Table 8-1]

[0216] [Table 8-2]

[0217] Detailed ophthalmological examination of a PSEN1 E280A mutation carrier with two APOE3ch alleles An evaluation was conducted. This holder had a visual acuity of 20 / 70 in the right eye and 2 in the left eye. The patient had a visual acuity of 0 / 40. Anterior segment examination revealed a posterior chamber lenticular The anterior segment of the left eye showed significant nuclear sclerosing cataracts (Figures 4A and 4B). ). Examination of the posterior segments of both eyes was normal, with a clear vitreous cavity, normal-looking optic nerve, macula, and The patient had peripheral retina. Further examination of the right eye using optical coherence tomography (OCT) revealed the presence of the fovea. Except for increased reflexes in a small area covering it, it was normal (Figure 4D). Figure 4C shows the same as in Figure 4D. The image shows an infrared image of the right eye depicting a cross-section of the retina (lines). Furthermore, the OCT image of the left eye is shown. This results in small defects in the outer boundary layer and ellipsoidal layer (arrows in Figure 4F), Degenerated laminar fissures (indicated by "*" in Figure 4F) were revealed.

[0218] Several mechanisms have been proposed to explain the impact of the APOE variant on AD risk. However, most studies focus on Aβ 42 Their differential effects on aggregation and plaque volume (Concentrated in APOE2<3<4) 47 In this study, regarding the clinical onset of AD The resistance of the participants was a) longer than 70 years. 42 Despite overproduction, relatively low levels a) Is it related to the amount of Aβ plaque, or a) is it related to a relatively high amount of Aβ plaque? , Paired spiral fiber (PHF) tau (amount of nerve fiber change) and limited downstream measurement of neurodegeneration Neuroimaging measurements were used to determine if the values ​​were correlated with constant values.

[0219] The participants' neuroimaging findings are shown in Figure 5. Positron emission tomography (PE) T) The images are superimposed on the medial and lateral surfaces of the left hemisphere. The top row shows amyloid plaques. The PET measurement values ​​(PiB DVR) are shown. The bottom row shows the paired helical fiber (PHF) tau. This shows the PET measurement of the amount of (i.e., neurofibrillary tangles). People with late-onset MCI have a PET measurement of 70. For those in their 20s, the typical age at which MCI develops is 44 years old.

[0220] As shown in Figure 5, individuals with late-onset MCI developed MCI in their 40s. Among PSEN1 E280A owners, a higher average (DVR of 1.49-1.60) Ratio of Pittsburgh Compound B (PiB) distribution volume in the cortex relative to the cerebellum As shown by (DVR=1.96), PET measurements with an abnormally high amount of Aβ plaque She had a stable value. Despite her high Aβ plaque volume, the magnitude of her PHF tau volume was The spatial extent and neurodegeneration were relatively limited; her Flortausippi Tau PET measurements are limited to the medial temporal lobe and not very extensive, but also include the occipital region. While affected, it was relatively less affected in other areas characteristic of the clinical stages of AD. Figure 5). Her fluorodeoxyglucose PET measurement of brain metabolic rate for glucose. The values ​​are conserved in brain regions known to be preferentially affected by AD, and are more youthful. Individuals with the PSEN1 E280A mutation who developed MCI at an early age and many younger, recognized cases In mutants whose abilities were not impaired, higher whole-brain precuneus measurements were included. .

[0221] Figure 6 shows the mean cortical amyloid plaque volume, entorhinal cortex PHF tau volume, hippocampal volume, and precuneus volume. The glucose metabolism measurements are shown. These measurements are based on two APOE3ch alleles and A PSEN1 E280A mutation carrier with an exceptionally late onset of MCI (red dot), M PSEN1 E28 with MCI at a younger age than typical for the family at the time of CI onset. 0A mutation carriers (black dots) and PSEN1 E280 individuals who have not yet developed MCI. Based on brain imaging results obtained from A mutation carriers (gray dots). Amyloid plaque amount. This is expressed as the ratio of cortical distribution volume to mean cerebellar volume (DVR). The amount of fiber (PHF) tau is the standard uptake value of flortaucipillin (FTP) in the entorhinal cortex relative to the cerebellum. It is expressed as a ratio (SUVR). Hippocampal volume, which can decrease due to hippocampal atrophy, is relative to the whole brain. It is expressed as a volume ratio of the hippocampus. Affected by AD, which involves synaptic dysfunction and loss. Brain glucose metabolism, which is reduced in the affected brain regions, is lower in the precuneus compared to the whole brain. This is reflected as the brain metabolic rate (CMRgl) ratio for S. As shown in Figure 6, Individuals carrying the PSEN1 E280A mutation with two APOE3ch alleles are far more likely to be affected. Although she also had a high amount of amyloid plaques, she also had comparable severe PHF tau levels and hippocampal atrophy. There was no evidence of decreased glucose metabolism in the precuneal region. AD and / or normal aging. Her MRI-based measurement of the hippocampus relative to the whole brain is a measure of hippocampal atrophy that can be affected by the disease. The volume was within the range of mutation carriers who developed MCI in their 40s. (The theory was constrained) I don't intend to, but these results suggest that the clinical development of AD in this APOE3ch homozygous individual is not related to the clinical manifestations of the disease. Resistance to this limits tau pathology and neurodegeneration even when faced with high Aβ plaque levels. This suggests that it is mediated through a mechanism.

[0222] To study the functional effects of APOE3ch variants, we used bacterial-derived wild-type human APOE3ch variants. In the presence of E3 protein, in the presence of mutant ApoE3ch protein, or any A In vitro Aβ in the absence of poE protein 42 Aggregation was compared. Aβ 42 fiber The rate of formation was detected by thioflavin T fluorescence. Aβ 42 Aggregation is caused by wild-type human Apo The highest level was observed in the presence of the E3 protein (C-terminal domain), and in the presence of human ApoE3ch. Lower (observed in the presence of ApoE) 48 (Similar to) Any ApoE thing The lowest level was observed in the absence of the substance (Figure 7).

[0223] This finding was confirmed using a highly sensitive split ciferase complementation assay, and In the assay, the luciferase signal is detected once amyloid is most toxic. It is a type of amyloid 49 If oligomers are formed, they are reconstructed. 48 Oligo Luciferase luminescence due to merm formation is more pronounced in ApoE3ch compared to wild-type ApoE3. As shown in Figure 8, full-length ApoE3ch expression in mammalian cells was significantly reduced. , significantly lower Aβ compared to wild-type ApoE3 42 This caused oligomerization. These results provide confirmation of the genetic analysis, and the protective effect of the ApoEch protein is At least in part, Aβ 42 This suggests that it may be due to its limited ability to promote aggregation. This study found that participants without the APOEch / 3ch genotype lived to be in their 70s. If present, it is possible that there would have been an even higher level of Aβ plaque deposition, and The ApoE3ch protein changes the morphology of Aβ aggregates, leading to downstream neuroinflammation, tau pathology, and neurological changes. It is still conceivable that the changes were made in a manner that limits sexual and cognitive decline. ru.

[0224] This includes four individuals with the PSEN1 E280A mutation who progressed to MCI at a median age of 45. Hmm, a small number of Colombian family members have one copy of the APOE3ch mutation. A solution was found. 50 The reason for this is that APOE3ch homozygosity is autosomal It was hypothesized that this would dramatically reduce the risk of overt AD and delay its clinical onset. Because it was small, APOEch heterozygotes were less likely to exhibit cognitive decline associated with autosomal dominant AD. Partial protection and substantial protection against sporadic, late-onset AD and / or neurodegeneration. It is still conceivable that it may have

[0225] These results indicate that APOE variants differ in the degree of their pathogenicity (AP OEch and APOE2<3<4), APOE3ch / 3ch and APOE2 / 2 are This suggests that it is associated with the greatest loss of function. APOE should be safely and adequately edited. Interventions that collect, reduce its expression, or inhibit its pathogenic function are treatments for AD. This could have a significant impact on preventing infection. Interestingly, antiseptics in Aβ-overproducing mice... Suppression of APOE expression in the brain using lance oligonucleotides leads to changes in Aβ plaque morphology. and resulted in fewer degenerated neurites. 51 This approach is feasible. The lack of APOE expression is associated with middle-aged men who are homozygous for the frameshift variant. It is tolerable in 52 Statins can be used to treat HPL-III. This is because it supports the potential tolerance of ApoE reduction treatments. For example, Reiman et al. Nat See Commun 1191):667, 2020.

[0226] I don't intend to be bound by theory, but these results are about APOE3ch Mozygosity and APOE2 are associated with high resistance to the clinical onset of Alzheimer's disease. This means that these genotypes directly or indirectly limit downstream tau pathology and neurodegeneration. By doing so, these beneficial effects will be exerted; and these effects will be ApoE Despite the relative decrease in mediated Aβ aggregation, it is solely due to the size of the Aβ plaque. This further suggests that it is not based on these findings. These findings affect the understanding and treatment of AD. This suggests the role of APOE in prevention and this failure. This could stimulate interest in developing genetic and drug therapies to modify the condition.

[0227] [Example 2] Heparin-binding properties of APOE3ch mutant proteins material and method Heparin column protocols: ApoE2, ApoE3, ApoE3ch, and A The heparin binding affinity of the poE4 protein isoform was measured using a 1 ml heparin column (B The comparison was performed using ioVision-6554-1). The columns were cooled for 1 hour before use in the room. The column was allowed to acclimate to the temperature. The column was washed with 5 mL of 20 mM TRIS-HCl (pH 7.5). Then, 50 μg / mL of APOE compound in 20 mM TRIS-HCl (pH 7.5) was used. A 1 mL sample containing replacement protein was passed through the column five times for recirculation. Then, The column was washed five times with 20 mM TRIS-HCl (pH 7.5). The increasing NaCl gradient (0.025~1M) in mM TRIS-HCl was passed through the column. Then, a 1 mL fraction was collected and subsequently prepared for Western blotting.

[0228] Western blotting: Western blotting is performed using a heparin-conjugated column. Elution of ApoE isoform was confirmed in the collected fraction. The fraction was then analyzed using 10 μl of RI. PA buffer (Cell Signaling Technology), 4 μl D Dilute TT (1M) and 10 μl Laemmli buffer to a final volume of 40 μl. The sample was processed using 4-20% Mini-PROTEAN(registered trademark) TGX(trademark) powder. Separation on a recast protein gel (Bio-Rad) and nitrocellulose membrane (VW Transfer to R; 27376-991) and Odyssey blocking buffer (LI- Blocking was performed using COR Biosciences (Lincoln, NE), and mouse antibodies. his tag (Novus Biologicals.) antibody and IRDye 800C The immunoreactivity was investigated using W donkey anti-rabbit antibody (LI-COR Biosciences). The bands were visualized using the Odyssey infrared imaging system, and the image was taken. Visualization was performed on Studio version 2.1 (LI-COR Biosciences). The individual gels were then combined to create Figure 10.

[0229] Heparin plate ELISA protocol: ELISA is performed using heparin microplates. This was performed using Bioworld (50-197-531). These were then used for sample preparation. The cells were blocked for 1 hour using reagent (DY008). The heparin plates were then recombinant. ApoE protein isoforms (ApoE2, ApoE3, ApoE3ch, and Incubate each well with 0.1 μg / well of ApoE4 for 2 hours, then p The rate was washed five times in PBS containing a NaCl gradient (0-0.5M), and then... The sample was washed three times in washing buffer (DY008). Anti-His tag antibody (Novus biologicals; NBP2-61482) was mixed overnight at a 1:10,000 dilution. The plate was then refrozen for 5 minutes to ensure the removal of unbound primary antibodies. Wash several times, incubate with donkey anti-rabbit HRP (1:10000) for 45 minutes, The sample was washed five times to ensure the removal of secondary antibodies. ELISA reagent kit (DY008) Add sulfuric acid from the start of the reaction detection step, 100 μl of tetramethylbenzidine (Mil The mixture was heated to 37°C before adding lipore. After incubation for 5 minutes, sulfuric acid was added. Then, the reaction was terminated. After that, the plate was SPECTRAmax plus 384 The readings were obtained using molecular devices. The reading wavelength was 450°. The value was nm. Serially diluted recombinant Not Based on the ch3 protein, Prism 6 (GraphPad Software) The standard curve was plotted using [this method].

[0230] result The heparin sulfate proteoglycan (HSPG) portion is responsible for the plasma membrane and extracellular matrix. It is a type of glycosaminoglycan found within hundreds of proteins located in the middle. SPG-mediated protein-protein interactions are involved in amyloid and tau diseases. It plays an important role in numerous processes related to the pathogenesis of Alzheimer's disease, including neurodegeneration. It plays a role. Various ApoE isoforms, including ApoEch, heparin (HSPG) - Glycosaminoglycans are commonly used to model protein-protein interactions. The ability to bind with was investigated. In short, under an increasing NaCl gradient (0~0.65M) Includes ApoE isoforms, ApoE2, and ApoE4 eluted from a heparin column. The fractions were analyzed using ELISA. As shown in Figure 9, Alzheimer's ApoE variant ApoE4, associated with a higher risk of the disease, is protective. Compared to the known variant ApoE2, it has a higher affinity for heparin. Next, H eluted from the heparin column under an increasing NaCl gradient (0-0.65M). Images containing the tags ApoE2, ApoE3, ApoE4, and ApoE3ch The minutes were analyzed using Western blotting. As shown in Figure 10, ApoE3 ch has impaired heparin binding, and it is much lower than that of ApoE2. The affinity of the ApoE isoform to the materials is also described in the materials and methods section. The analysis was performed using a heparin plate ELISA protocol, as shown in Figures 11A-11B. As has been shown, ApoE3ch is required for ApoE4 release compared to what is needed. At much lower concentrations of NaCl, ApoE3ch was released from the heparin column. It showed remarkably low levels of heparin binding.

[0231] [Example 3] Production of antibodies against wild-type ApoE and ApoEch mutant proteins material and method Antibody competition assay: Antibody is used to test ApoE3 recombinant protein (20 mM Tris-H Incubate in CL at a 1:10 ratio (50 ug / ml) and at room temperature for 3 hours. The following were included: a negative control containing only the culture medium and a control containing only the recombinant protein ApoE3. A positive control was used. The antibody / ApoE3 recombinant protein solution and the control were analyzed using a heparin column. It was passed through and exposed to an increasing NaCl gradient (as described in Example 2 above). The fractions were collected and evaluated by ELISA and Western blotting.

[0232] BCA assay: The fraction collected from the heparin column is first analyzed using bicinchoninate assay. Using the BCA assay (Pierce BCA protein assay kit) Cleaned. The assay was performed using 200 μl of reagent A and B mix and 25 μl of reagent B mix. The experiment was conducted using each of the fractions. The 96-well plates were incubated at 37°C for 30 minutes. The reading was taken at 562nm. The plate was read using a Synery 2 microplate reader. (BioTek Instrument Inc.) and Gen5 version 1.11 software It was read using software.

[0233] Western blotting: Western blotting is performed using a heparin-conjugated column. The elution of recombinant ApoE3 protein in the collected fraction was confirmed. The fraction was then processed using RIPA. buffer (Cell Signaling Technology), 10×(DTT 1M) and 40 μl final volume in 10 μl using 4×Laemmli buffer The sample was diluted as follows: 4-20% Mini-PROTEAN(registered trademark) TGX( Separation on a precast protein gel (Bio-Rad) and nitrocellulose (trademark) Transfer to a membrane (VWR; 27376-991) and use Odyssey blocking buffer ( Blocked by LI-COR Biosciences (Lincoln, NE), Us anti-his tag (Novus Biologicals.) antibody and IRDye 8 The search was conducted using 00CW donkey anti-rabbit antibody (LI-COR Biosciences). The reactive band was visualized using the Odyssey infrared imaging system, and Ima Compatible with GE Studio version 2.1 (LI-COR Biosciences). It was visualized.

[0234] ELISA: An antibody designed against the heparin-binding domain of ApoE is used to target ApoE. Regarding their affinity for E3 and ApoEch mutant recombinant proteins, ELI The test was performed using SA. The Ni-NTA HisSorb plate (Qiagen) was washed. Washed three times with purification buffer 1 (DY008). ApoE recombinant protein was used. The solution was suspended in buffer (DY008) to achieve a final concentration of ug / ml. The plate was then... Incubate with 200 μl of ApoE recombinant protein for 2 hours, then wash with a 1× wash buff. I washed it 5 times with AA (DY008). After that, I washed the plate from 1:1,000 to 1:3 Antibodies were incubated overnight at 4°C in serial dilutions up to 2,000. Then, the solution was applied. Wash the tortoise five times with 1x washing buffer (DY008) and then use anti-mouse HRP (Abcam). Incubate with (ab97046, 1:10,000) for 45 minutes, then bind To ensure complete removal of any remaining secondary antibodies, the tank was washed five times in 1x wash buffer. Sulfuric acid from the ELISA reagent kit (DY008) was warmed to 37°C. 100 μl of tetraphosphate was added. The reaction detection step was initiated by adding methylbenzidine (Millipore). 5 After incubation for several minutes, sulfuric acid was added to terminate the reaction. Then, the plate was removed. Synery 2 Microplate Reader (BioTek Instruments.I The data was read using nc) and Gen5 version 1.11 software.

[0235] result Monoclonal antibodies were created against amino acids 130-143 of ApoE, and full-length ApoE antibodies were produced. The effect of this study on the binding between the oE3 protein and heparin was investigated. Briefly described... If so, the full-length wild-type ApoE3 protein or the monoclonal antibody and pre-incubate The baited molecules are then passed through a heparin column to ensure maximum ApoE3 binding. The column was recycled five times. Afterward, the column was treated with 20 mM Tris-HCl (pH=7). 5) Wash 5 times and observe the increase in NaCl in 20 mM Tris-HCl (pH=7.5). Exposure to a temperature gradient (0-1M). Elution from Tris-HCl washing and various NaCl Elution was collected based on concentration (Figures 12A-12C).

[0236] The fractions collected from the column are first screened using a bicinchoninic acid (BCA) assay. The analysis was performed. As shown in Figure 13A, for wild-type ApoE3 in the fraction, The protein signal is detected on the right side of the curve, indicating strong binding of ApoE3 to heparin. In contrast, when ApoE3 is pre-incubated with a monoclonal antibody... A strong signal was observed in the initial fraction at low ion intensity (Figure 13B). To verify the results, Western blotting was used to collect samples from a heparin column. The column wash solution and NaCl gradient fraction were analyzed. As shown in Figure 14, A monoclonal antibody against live ApoE3 (A3Ab) (This antibody is from Arboleda-Velasquez e (In t al., Nature Medicine, 25, pages 1680-1683 (2019), it was named 1343.) Pre-incubation with the ApoE3ch mutation enhances its ability to bind to heparin. The levels were reduced to a level similar to that of body proteins. The individual gels were then combined to create Figure 14. This was achieved. These results indicate that the antibody modified the binding properties of ApoE to heparin, and thereafter... to prevent or treat Alzheimer's disease or related dementia or neurodegeneration. This suggests that it can be used for that purpose.

[0237] To produce a monoclonal antibody against the heparin-binding domain of ApoE, The wild-type ApoE peptide: KLH-CTEELRVRLASHLRK-CONH 2 (SEQ ID NO: 54) and ApoEch peptide: KLH-CTEELRVSLASHL Immunization was performed using RK-CONH2 (SEQ ID NO: 55). The conjugation of the peptide... A cysteine ​​residue was added to the N-terminus to promote cell fusion. The cell supernatant was then used to assess the activity of wild-type and mutant peptides and proteins. The tests were conducted. The seven antibodies produced were, as an example, described in the Materials and Methods section. As shown, the analysis was performed by ELISA. The 19G10-2 antibody serum showed APOE3 channel mutation. Specificity to both the full length and C-terminus of isoproteins, and wild-type APOE3 protein It showed some interaction with quality (Figure 15A). The 23B2 antibody and wild-type ApoE3 Both C-terminal and full-length recombinant proteins of the ApoE3ch mutant were shown to be receptive to recombinant proteins of their respective C-terminuses. (Figure 15B). The 2H79-1 antibody exhibits nonspecific binding to bovine serum albumin (BSA). It showed no affinity for either wild-type ApoE3 or ApoE3ch mutants. (Figure 15C). Both the 30E1-2 antibody and the 16H8 antibody showed full length and C-terminal Ap The oE3ch mutant protein and wild-type ApoE3 demonstrated reactivity to the C-terminal form. However, it did not react with the full-length wild-type APOE3 protein (Figures 15D and 15E). 2 5F1-2 antibody serum showed high resistance to full-length and C-terminal ApoE3ch mutant proteins. It exhibits high affinity, variable binding to the C-terminus of the wild-type ApoE3 protein, and full-length wild-type binding. It appeared to have some interaction with the ApoE3 protein (Figure 15F). The 29G10-2 antibody targets both the full length and C-terminus of the ApoE3ch mutant protein. It showed high affinity and also demonstrated reactivity to C-terminal wild-type ApoE3 and BSA. The 29G10-2 antibody did not interact with the full-length wild-type ApoE3 protein (Figure). 15G).

[0238] Variable heavy chain (VH), variable light chain (VL), and The sequences of the complementarity-determining regions (CDRs) are described herein.

[0239] Furthermore, the following parental clones possessing specificity for wild-type ApoE were created, and wild-type A poE peptide (WT peptide), wild-type ApoE protein (WT protein), mutation Mutated ApoE peptide (ApoEch; Mut peptide), and mutant ApoE3ch The specificity for the protein (Mut protein) was tested as shown in Table 6. The value indicates the absorbance level when detected by ELISA. The peptide is wild-type ApoE peptide (KLH-CTEELRVRLASHLRK-CONH It demonstrated specificity for protein 2 (SEQ ID NO: 54) and wild-type ApoE protein.

[0240] [Table 9]

[0241] [Example 4] Production of a fusion protein containing the heparin-binding domain of APOE material and method Peptide competition assay: Wild-type ApoE3 and ApoE3ch mutant peptides (5 (0 ug / ml) ApoE3 recombinant protein (prepared in 20 mM Tris-HCl) The solution was incubated with 50 ug / ml at room temperature for 3 hours. Then, the peptide / Ap The oE3 recombinant protein solution was passed through a heparin column and exposed to an increasing NaCl gradient. (As described in Examples 2 and 3 above). Collect the fractions and Western blot. Evaluation was performed using a lottery system.

[0242] Western blotting: Western blotting is performed using a heparin-conjugated column. The elution of ApoE3 in the collected fraction was confirmed. The fraction was then mixed with 10 μl of RIPA buffer. -(Cell Signaling Technology), 10×(DTT 1M) The sample was then diluted to a final volume of 40 μl in 4×Laemmli buffer. ~20% Mini-PROTEAN(registered trademark) TGX(trademark) precast protein The samples were separated on a cellulose gel (Bio-Rad) and then separated into a nitrocellulose membrane (VWR;27376-9 Transfer to 91) and Odyssey Blocking Buffer (LI-COR Biosci Blocking with ences, Lincoln, NE), and mouse anti-his tag (Novu s biologicals) antibody and IRDye 800CW donkey anti-rabbit (LI- The immunoreactivity band was searched using antibodies from COR Biosciences. Visualization was performed using an infrared imaging system, and the Image Studio version... Visualization was performed using 2.1 (LI-COR Biosciences).

[0243] result Amino acids 130-1 of wild-type ApoE protein and ApoEch mutant protein Peptides containing 43 were each prepared. The wild-type ApoE3 group of these peptides was also analyzed. To investigate the effect on the binding between replacement protein and heparin Sepharose, the materials and A peptide competitive assay was performed as described in the method. This is shown in Figure 16. Thus, the wild-type ApoE peptide is a fraction of the wild-type ApoE3 recombinant protein-bound peptide. This peptide causes a fuzzy reaction, and this peptide competes with wild-type full-length ApoE3 for binding to heparin. These results suggest that amino acid 13 of the wild-type ApoE protein is possible. ApoE fragments containing 0-143 alter the binding properties of ApoE to heparin, This helps prevent or treat Alzheimer's disease or related dementia or neurodegeneration. This suggests that it can be used for that purpose.

[0244] To increase the protein stability of the aforementioned peptide, human ApoE (wild type and R The heparin-binding domain or heparin-binding domain of the 136S mutant version C-terminal and N-terminal fusion proteins containing allosteric regulatory sites are backbone Fc IgG2 constructs pfuse-hfc1 and pfcn-hg2 (In vivog The human ApoE fragments were prepared using (en). The human ApoE fragments are APOE2 and APOE4 variants. The site in question was excluded. Administration of the ApoE fragment having R at position 136 was used to treat HSPG. In interactions with binding partners, including endogenous ApoE, it competes with ApoE for protection against neurodegeneration. This may result in a binding of the ApoE fragment with S at position 136 to wild-type ApoE. By binding incompatible molecules, it may be possible to provide protection against neurodegeneration. This involves creating fusion proteins. amino acids from fragments of wild-type and R136S mutant ApoE used for this purpose. The acid sequence is shown below (position R136 is shown in bold and double-underlined). (ru). Downstream of the R136 fragment WT STEELRVRLASHLRKLRKRLLRDADDLQK (Sequence ID 57) mutant STEELRVSLASHLRKLRKRLLRDADDLQK (Sequence ID 58) Upstream of the R136 fragment WT RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (Sequence ID 59) mutant RLVQYRGEVQAMLGQSTEELRVSLASHLRKL(Sequence ID 60)

[0245] Figures 17A-17D show a model of the interaction between the ApoE fragment and heparin. Figure 17A This is the wild-type ApoE fragment (downstream of the R136 fragment, helix) that interacts with heparin. The model is shown. Figure 17B shows a fragment of ApoE R136S interacting with heparin (R1 Figure 17C shows a model of the downstream helix of 36 fragments. A model of the bio-type ApoE fragment (upstream of the R136 fragment, helix) is shown. Figure 17D shows the helix. A fragment of ApoE R136S that interacts with parin (upstream of the R136 fragment, helix) The model is shown below.

[0246] Exemplary fusions containing either a downstream or upstream component of the R136 fragment. The nucleic acid and amino acid sequences for the protein constructs are shown below. 184Q pfcn-hg2 ApoE 114~144 Nucleic acid sequence (SEQ ID NO: 81)

[0247] [ka]

[0248] [ka] Amino acid sequence (SEQ ID NO: 82)

[0249] [ka] 184R pfcn-hg2 ApoE 114~144 R136S Nucleic acid sequence (SEQ ID NO: 83)

[0250] [ka]

[0251] [ka] Amino acid sequence (SEQ ID NO: 84)

[0252] [ka] 197F pfuse hfc2 ApoE 114~144 Nucleic acid sequence (SEQ ID NO: 85)

[0253] [ka]

[0254] [ka] Amino acid sequence (SEQ ID NO: 86)

[0255] [ka] 197G pfuse-hfc2 ApoE 114~144 R136S Nucleic acid sequence (SEQ ID NO: 87)

[0256] [ka]

[0257] [ka] Amino acid sequence (SEQ ID NO: 88)

[0258] [ka] 184U pfcn hg2 ApoE 129~157 Nucleic acid sequence (SEQ ID NO: 89)

[0259] [ka]

[0260] [ka] Amino acid sequence (SEQ ID NO: 90)

[0261] [ka] 184V pfcn hg2 129~157 R136S Nucleic acid sequence (SEQ ID NO: 91)

[0262] [ka]

[0263] [ka] Amino acid sequence (SEQ ID NO: 92)

[0264] [ka] 197H pfuse hfc2 ApoE 129~157 Nucleic acid sequence (SEQ ID NO: 93)

[0265] [ka]

[0266] [ka] Amino acid sequence (SEQ ID NO: 94)

[0267] [ka] 197I pfuse hfc2 ApoE 129~157 R136S Nucleic acid sequence (SEQ ID NO: 95)

[0268] [ka]

[0269] [ka] Amino acid sequence (SEQ ID NO: 96)

[0270] [ka]

[0271] [Example 5] CRISPR-Cas9-mediated editing and base editing of APOE To introduce the R136S mutation into APOE using CRISPR-Cas9, gRN Sequence A was designed (Table 7). In one example, the gRNA sequence is downstream of the U6 promoter. To form a linker containing a 20-base sequence to be cloned into the BsmB1 site. It is cloned into lentiCRISPR v2 using two oligonucleotides. To support this, we designed a template with two additional silent mutations. The template is the same as the aforementioned mutations. It has 50 base pairs adjacent to the area with the difference. An exemplary template sequence is as follows:

[0272] [ka] However, silent mutations that disable the PAM motif are double-underlined and R13 The codon corresponding to the 6S mutation is shown in bold, and is from the PCR product of the template-accepting clone. Silent mutations that generate the SacI site for cleavage are shown in italics. It is.

[0273] [Table 10]

[0274] I don't intend to be bound by theory, but here is a proposed mechanism for APOE3ch mutations. This is loss of function (in binding with HSPG). Therefore, the gRNA sequence is CR The system was designed to "knock out" APOE using ISPR-Cas9 (Table 8). The gRNA was designed to target exon 3 (amino acids 1-61) of ApoE. In this example, the gRNA sequence is cloned to the BsmB1 site downstream of the U6 promoter. Two oligos are ordered to form a linker containing the sequence of 20 bases to be linked. By doing so, it is cloned into lentiCRISPR v2. Repair is non-homologous This is done by end joining (NHEJ), which is an error-prone method and often... This can result in short insertions or deletions that lead to APOE knockout.

[0275] [Table 11] R136H mutations in APOE are predicted to produce effects similar to those of R136S mutations. Therefore, in order to introduce the R136H mutation into APOE using base editing technology, The following gRNA was designed (Table 9). gRNA sequence: GAGGCGCACCCGCAGCTC CT is a 20-base sequence cloned into the BsmB1 region downstream of the U6 promoter. Using two oligonucleotides to form a linker containing pLenti sgRNA Cloned into (addgene 71409). Plasmid Addgene base The editor plasmid pCMV-BE3 (#73021) is used to induce base editing. It is used.

[0276] [Table 12]

[0277] [Example 6] High-throughput screening of molecules that modify the binding of ApoE to heparin To screen for molecules that affect the binding of ApoE to heparin, A PoE proteins can be used with candidate polypeptides, small molecules, nucleic acids, lipids, or carbohydrates and plain nucleic acids. Incubate. Heparin-coated the pre-incubated ApoE protein. It is introduced onto a chemical surface (such as a plate or column) and binds to heparin / HSPG / GAG. This allows the unbound ApoE and candidate molecules to be washed away. Alternatively, heparinco Before applying the ApoE protein to the coated surface (such as a plate), remove the candidate protein. It can be pre-incubated with offspring. The level of ApoE bound to heparin is reduced against Apoe / heparin was detected using body, protein assay, or fluorescence assays. Evaluate the effect on binding. Candidate molecules that reduce ApoE / heparin binding are used in dementia and and / or measures for the prevention or treatment of cognitive decline associated with mild cognitive impairment. This could potentially correspond to a novel therapeutic substance for humans who need it. Examples of such molecules include EZ-482 (for example, Mondal et al. Biochemistry 55(18):2613-21). See 2016. The structure of the EZ-482 is shown below.

[0278] [ka]

[0279] [Example 7] High-throughput screening of molecules that modify the binding of ApoE to anti-ApoE antibodies. To screen for molecules that affect the binding of ApoE to anti-ApoE antibodies. The ApoE protein is pre-treated with candidate polypeptides, small molecules, nucleic acids, lipids, or carbohydrates. Incubate. Pre-incubate the ApoE protein into the ApoE HS. An antibody that binds to the PG binding site (any anti-ApoE antibody as described herein) The antibody is introduced onto a surface coated with (a) and bound to it. This allows the unbound ApoE and candidate molecules to be washed away. Alternatively, heparin Before applying the ApoE protein to a coated surface (such as a plate), It can be pre-incubated with comolecules. The bell is detected using an antibody, protein assay, or fluorescence assay, and the binding of the candidate molecule is determined. Evaluate the effect on cognitive function. Candidate molecules that increase or decrease ApoE / heparin binding are considered to be cognitive functions. For the prevention or treatment of cognitive decline associated with the disease and / or mild cognitive impairment, such This could potentially correspond to a novel therapeutic substance for human subjects requiring such treatment.

[0280] [Example 8] Antibody characterization Furthermore, antibodies that bind to wild-type ApoE and ApoEch variants are also available in heparin-based formulations. T-chromatography, Western blotting, subclonal analysis, monoclonal Antibody screening, screening for selectivity between huApoE3 and msApoE Evaluation was performed using anesthetic and in vivo subretinal injections.

[0281] method Chromatography experiment His-tagged recombinant ApoE peptide (50 μg / mL) was administered to 20 mM T Each antibody was diluted 1:10 in ris HCl buffer (pH 7.5) and incubated at room temperature for 3 hours. The sample was incubated. The heparin binding of the sample was determined using a heparin Sepharose column. The test was conducted by allowing the column to reach room temperature and using 20 mM Tris- Washed 5 times with HCl. 10 μL was collected for the WB experiment. The input was loaded onto the column. The input was recycled through the column five times. The passing fraction is collected, then the column is washed five times, and the recovered fraction is called the "washing solution". Labeled. 1 mL fractions were labeled in 0.05 M steps of the NaCl salt gradient from 0 M to 1 M. A 1 mL fraction was collected. A 5 M fraction was used to ensure complete release of the protein from the column. The minutes were also tested. Using WB or ELISA, the binding of ApoE to heparin was tested. We tested the changes.

[0282] Western blotting For WB analysis, prepare the sample in 4× sample buffer (Laemmli's SDS sample). Buffer (BP-110R, Boston Bioproducts), 4 μL DT T (Sigma Aldrich) and each in 16 μL 1× RIPA buffer The fraction was prepared by diluting 10 μL of the fraction. Electrophoresis was performed using vertical electrophoresis under denaturing conditions. The procedure was performed using a chamber (Biorad). The bands were formed using 4-20% precast gel. The data was separated using a constant voltage (70V for 15 minutes, 100V for 1 hour) on Biorad. Protein transcription is performed on a nitrocellulose membrane (Millipore) at a constant voltage of 70V. The procedure was performed for 1 hour. The membrane was treated with Odyssey Blocking Buffer (Licor). Block at room temperature for 1 hour, then remove the membrane with primary antibody (anti-His, rb (rabbit), 1:5 000, Novus biological) and secondary antibody (Roba anti-rb-800, 1 :10000, Licor) Before incubation, TBS-0.05%Twee The samples were washed with a 20°C (Thermo fisher) for 3 x 10 minutes. Images were acquired using Od. The scan was performed using a Yssey scanner. The data was analyzed using ImageJ. The data is standardized by the input, and the fraction number (0 = input, 1-27 = 20mM T) is used. Ris HCl at pH 7.5 shows increasing step gradient of 0.05M NaCl, 28 = 20mM The standardized intensity against 5M NaCl in TrisHCl (pH 7.5) was expressed as the standardized strength.

[0283] ELISA Selectivity of antibodies against huApoE3WT, huApoE3ch, or msApoE To test this, a plate coated with anti-his ELISA was used, 0.002 Incubate with 5 μg / μL of target protein at room temperature for 2 hours with gentle shaking. The wells were washed five times with 1x washing buffer (R&D), and then the wells of interest were... Serial dilutions of the Innovagen antibody were incubated overnight at 4°C on a shaker. (100 μL / well). The next day, wash the wells five times with sample buffer and feed to rabbits. Anti-mouse HRP conjugated buffer (1:10,000; 100 μL / well; The plates were incubated for 45 minutes using Abcam. After five more washes, the plates were then removed. The colorimetric reaction was started by incubating with pigment source A + B in a 1:1 ratio (100 μL / wet). The reaction was stopped with 2N sulfuric acid (R&D stop solution, 50 μL / well), and the absorbance was measured. It was detected spectroscopically at 450 nm.

[0284] Antibodies designed against the heparin-binding domain of APOE, APOE3 and AP The affinity of the OEch mutant for recombinant protein was tested using ELISA. Wash Ni-NTA HisSorb plate (Qiagen) with washing buffer 1 (DY0 Wash three times with 08) to a final concentration of 0.5 ug / ml of APOE recombinant protein. The solution was suspended in buffer (DY008). The plate was then soaked in 200 µl for 2 hours. The plates were incubated. Then, the plates were washed five times with 1x washing buffer (DY008). Cleanse the plate, then test it in a serial dilution series from 1:1000 to 1:32000. The body was incubated and incubated overnight at 4°C. Then the plate was washed 1x. The plates were washed five times in cleansing buffer (DY008). Afterward, the plates were treated with anti-mouse HR. P(Abcam;ab97046)(1:10000) was incubated with the sample for 45 minutes. Then, wash the plate 1× to ensure complete removal of any unbound secondary antibodies. Washed 5 times in purification buffer. Sulfuric acid from ELISA reagent kit (DY008) To initiate the detection step of the reaction, add 100 μl of tetramethylbenzidine (Millipore). The mixture was warmed to 37°C before adding the sulfuric acid. After incubation for 5 minutes, sulfuric acid was added to terminate the reaction. Then, the plate was loaded into the Synery 2 microplate reader (BioTe kInstrument.Inc) and Gen5 version 1.11 software I used it to read the text.

[0285] result Antibody 1H4 was analyzed using heparin affinity chromatography and Western blotting. ApoE3 was evaluated using a negative control (medium, blot above) or 1H4. Incubate with one of the blots (below) and analyze each fraction using heparin affinity chromatography. The samples were subjected to tography and Western blotting (Figure 18A). ApoE3 positive bacteria The arrow indicates an antibody that specifically detects the his tag of recombinant human APOE. It is detected using an anti-his tag (rb, 1:5,000). Figure 18B is shown in Figure 18A. As described, quantitative analysis of WB blotting bands detected by antibody anti-his tag. The results show the normalization. The intensity was standardized relative to the input. These results are for ApoE3 HEPA The binding to phosphorus decreased in the presence of the antibody, and the antibody 1H4 binds to heparin. This demonstrates competition with ApoE in this context. N=2 independent experiments.

[0286] Next, subclonal analysis of 1H4 serum was performed. Figures 19A-19D show ApoE3 W T full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein 1H4-2 blood was tested using protein (C) and ApoE3ch peptide (D). The ELISA results for Kiyoshi are shown. The results are obtained at 450 nm relative to the dilution factor of the tested serum. It is expressed as optical density. Figure 20 compares the results shown in Figures 19A to 19D. This is a diagram.

[0287] Figure 21 shows either human recombinant ApoE3 or mouse recombinant ApoE3 and ink. The typical ELISA profiles of serial dilutions of the tubulated antibody 1H4 are shown. The results showed that the antibody preferentially binds to human proteins but not to mice. This demonstrates that antibody 1H4 is selective for human ApoE. The result was obtained at 450 nm. The average optical density detected is shown as ±sem(n=2).

[0288] Next, the monoclonal 1H4 antibody was purified from the cloned hybridoma and ELIS It was submitted for evaluation as A. Figure 22 shows the results from the ELISA experiment.

[0289] Antibody 7C11 was analyzed using heparin affinity chromatography and Western blotting. Quantitative ELISA for chromatographic fractions and competition for binding analysis Evaluation was performed using ELISA.

[0290] The heparin affinity chromatography fraction of ApoE3 was used as a negative control (medium, above Incubate with either the blot or antibody 7C11 (bottom blot), and The samples were subjected to stun blotting (Figure 23A). The ApoE3-positive band is indicated by the arrow. Furthermore, an antibody anti-his tag (rb, 1) specifically detects the his tag of the recombinant peptide. It is detected using (5,000). Figure 23B shows the antibody resistance as shown in Figure 23A. This shows the quantification of blotting bands in WB detected by the HIS tag. The intensity is measured in Standardized against the put. These results indicate that the binding of ApoE3 to heparin is the same as the aforementioned anti The decrease occurred in the presence of the body, and antibody 7C11 competed with ApoE for heparin binding. This indicates that they are combined.

[0291] Next, subclonal analysis of 7C11-1 serum was performed. Figures 24A-24D show ApoE 3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3 chain complete 7C11-1 serum using full-length protein (C) or ApoE3ch peptide (D) The ELISA results from the test are shown. The results are 450 for the dilution factor of the serum tested. This is expressed as optical density in nm. Figure 25 shows the results shown in Figures 19A-19D. This is a diagram comparing the two. Next, the monoclonal 7C11-1 antibody is applied to the cloned hybrid. The sample was purified and subjected to ELISA evaluation. Figure 26 shows the results from the ELISA experiment. .

[0292] Furthermore, the 19G10-2 antibody was subjected to heparin affinity chromatography, and Western chromatographs were also used. Chromoblotting, quantitative ELISA for chromatographic fractions, and binding Evaluation was performed using ELISA for analysis. ELISA screening of 19G10-2 antibody The HEP was used to compare recombinant proteins of APOE3 wild-type (WT) and APOE3ch mutants. The procedure was performed on the phosphorus-binding domain. As shown in Figure 27, the 19G10-2 antibody The body shows the full length of the APOE3ch mutant recombinant protein and the C-terminal domain (amino acid 12 Specificity for both 5-299), as well as some interaction with APOE3 WT. This indicates that.

[0293] Negative control (medium, upper blot) or antibody 19G10-2 (lower blot) Heparin affinity chromatography fraction of ApoE3 incubated with this. The samples were subjected to ELISA analysis (Figure 28A). The ApoE3 positive band is indicated by the arrow. Therefore, an antibody anti-his tag (rb, 1: It is detected using 5,000). Figure 28B shows the antibody anti-h as shown in Figure 28A. This shows the quantification of blotting bands in a WB (Wet Balance) detected by the is tag. The intensity is used as an input. Standardized for the set. These results are for the ApoE3ch-HSPG domain. Despite being designed to be compatible with wild-type ApoE in heparin binding, 19G10-2 is different. This indicates that competition occurred, resulting in a decrease in the binding of ApoE3 to heparin. I don't intend to be bound by theory, but the antibody 19G10-2 is heparin / HSPG APOE (e.g., APOE polymers or aggregates) that are less likely to bind / GAG It may be possible to recognize and / or stabilize structure-specific features.

[0294] Figure 29 shows ApoE3 WT vesicles incubated with 19G10-2 serum antibody. Tumblotting is shown. Top blot: Secondary antibody to detect 19G10-2 antibody. Membranes explored in mice. The membrane below was previously described for detecting the ApoE3-positive fraction. The antibodies were incubated with anti-his tags as shown. This analysis was performed on the antibody-APOE complex. (Left side of the blot; top and bottom) does not bind to heparin, while free APOE (blot) The right side (bottom) demonstrates high affinity binding to heparin.

[0295] Figure 30 shows serum 19G10-2 and mono for ApoE3 WT or ApoE3ch. A representative example showing the difference in binding of both clonal antibodies to hybridoma supernatant 19G10-2. This is an ELISA. The data shows the dominant selection of this antibody against the ApoE3ch variant. Selectivity has been confirmed. Figure 31 shows the antibody 19G10-2 (blood) in the presence of ApoE3 WT. Some limitations (clear, gray profile; monoclonal, black combined profile) This is a magnified view of the Y-axis showing the combined profile. Next, monoclonal 19G10-2 Antibodies were purified from cloned hybridomas and subjected to ELISA evaluation. Figure 32 shows EL The results from the ISA experiment are shown. The full length is higher than the recognition signal of the WT ApoE peptide. The recognition signal of WT APOE suggests the binding of structure-specific features. ru.

[0296] Furthermore, the 25F1-2 antibody was analyzed using heparin affinity chromatography and Western blotting. Quantitative ELISA and binding analysis of blotting, chromatographic fractions. Evaluation was performed using ELISA. Figure 33 shows APOE3 wild-type (WT) and AP 25F1-2 inhibitors of the heparin-binding domain of recombinant protein of the OE3ch mutant This shows ELISA screening of the body. As shown in Figure 33, 25F1-2 antibody It shows high affinity for the full-length and C-terminal proteins of the APOE3 mutant, but AP It does not appear to form a strong binding with the C-terminus of the OE3 WT protein, nor does it appear to form a strong binding with the APOE3 W protein. It showed a limited interaction with the full-length T protein. The results were obtained from the rare serum samples tested. This is shown as the optical density at 450 nm relative to the coefficient of variation.

[0297] Either a negative control (medium, upper blot) or antibody 25F1-2 (lower blot). The heparin affinity chromatography fraction of ApoE3 incubated with this was then used. The samples were then subjected to Western blotting (Figure 34A). The ApoE3-positive band was indicated by the arrow. This indicates that an antibody anti-his tag (r) specifically detects the his tag of the recombinant peptide. b, 1:5,000) is used for detection. Figure 34B is shown as in Figure 34A. This shows the quantification of blotting bands in WB detected by antibody anti-his tag. The input was standardized. These results are for the ApoE3ch-HSPG domain. Despite being designed for wild-type Ap, 25F1-2 has heparin binding. This indicates that it competes with oE, resulting in a decrease in the binding of ApoE3 to heparin. They are doing it.

[0298] Figure 35 shows ApoE3 WT incubated with 25F1-2 monoclonal antibody. Western blotting is shown. The blot above: secondary to detect 25F1-2. Membranes explored with anti-mouse markers. The membranes below were previously described to detect the ApoE3-positive fraction. The antibody-APOE complex was incubated with anti-his tags. This analysis was performed using an antibody-APOE complex. The body (left blot; top and bottom) does not bind to heparin, while free APOE does not bind to heparin. This demonstrates high affinity binding (right blot; bottom).

[0299] Figure 36 shows the results for 25F1-2 serum and monochloride in ApoE3 WT or ApoE3ch. This representative example shows the difference in binding between both 25F1-2 and 25F1-2 in the supernatant of ronal antibody hybridomas. This is an ELISA. These results indicate the dominance of this antibody against the ApoE3ch variant. Selectivity has been confirmed. Figure 37 is an enlarged view of the Y-axis of Figure 36, showing the presence of ApoE3 WT. The binding profile of antibody 25F1-2 under the following conditions is shown. Next, monoclonal 25F1- Two antibodies were purified from cloned hybridomas and subjected to ELISA evaluation. Figure 38 shows E The results from the LISA experiment are shown. The signal for recognition of the WT ApoE peptide is higher than that of complete Some recognition signals from long-WT APOE are due to the binding of structure-specific features. This suggests that...

[0300] Mouse antibody 1343ab (renamed from 23B2) was used in heparin affinity chromatography. Regarding tography and Western blotting, as well as chromatographic fractions The results were evaluated using quantitative ELISA. Figures 39 and 40 show the results for APOE wild-type (WT). and 13 for the heparin-binding domain of recombinant protein of APOE3ch mutant This shows ELISA screening of 43 antibodies. 1343 is APOE3 WT and APOE Both C-terminal and full-length recombinant APOE proteins of the 3ch mutants showed reactivity. (The mutant refers to the Christchurch mutant.)

[0301] Dissolve from heparin column using an increasing NaCl gradient in or without 1343 The ApoE in the resulting protein fraction was subjected to Western blotting (Figure 4). 1A). Individual blots were cut between 25kDa and 50kDa. Blank space The fraction separates individual blots. FT = Passing fraction. In the presence and absence of 1343, ELISA was performed to quantify the differences in NaCl elution patterns of different ApoEs. (Figure 41B). N=3 columns per isoform in independent experiments, and the difference was quantified. To simplify the analysis, the data was analyzed side-by-side twice on different days. The error bars indicate the standard error of the mean.

[0302] In the validation, mouse antibodies 1H4-2, 7C11-1, 19G10-2, and 25F1-2 were used. , and the CDR sequence derived from 1343ab, human IgG backbone (IgG2 or I The graft was applied to gG4) to create a humanized counterpart.

[0303] [Example 9] In vivo validation of ApoE antibodies We created an intraocular model of inducible APOE-dependent tau hyperphosphorylation (opposite helical fiber formation). Using this model, we investigated the effects of ApoE antibodies, which inhibit tau pathology, a marker of neurodegeneration. The abilities were tested. Figure 42A shows an exemplary experimental overview.

[0304] Briefly, his-tagged recombinant human ApoE3 was administered to B6;C3-Tg(Prn p-MAPT * P301S)PS19Vle / J mice (Jackson lab 00 8169) by intravitreal injection (Yoshiyama et al. 53(3):337-51, 2007). This mouse model carries the human tau P301S mutation and is a well-established animal model for Alzheimer's disease and other tauopathies such as frontotemporal lobar de mentia (see, e.g., Bugiani et al. 58(6):667-77, 1999). Mice injected with PBS were used as controls.

[0305] As shown in Figure 42B, in the control retinas of 6-week-old mice, phosphorylated tau paired helical filaments (PHF) were not present in ganglion cells and their axonal fibers (the signal inside the blood vessels labeled with isolectin B4 is a background signal). In contrast, administration of recombinant human ApoE3 (his-tagged) induced robust formation of PHF detected by the AT8 antibody. PHF were robust in ganglion cell axons (arrows) and ganglion cell bodies. Human ApoE was detected around ganglion cell bodies using an anti-His antibody.

[0306] Mouse 1H4-2 antibody and humanized 1343Ah antibody were injected intravitreally into the eyes of mice from the above mouse model (final volume 2 μL). The animals were sacrificed 3 days after injection, and the retinas were dissected and immunolabeled. The retinas were stained with DAPI, isolectin, and AT8 (pTAU) which recognizes phosphorylated paired helical filament tau (PHF tau). The retinas were processed with SP ​​​Images were obtained using a confocal microscope. See Figures 43B, 43C, 43F, and 43G. As shown, ApoE3 WT results in a significant increase in PHF tau, and this increase is , significantly reduced by 1H4-2 ( *** p<0.001). Similarly, humanized 1H 4-2 Administration of IgG2 / kappa recombinant monoclonal antibody is used in vivo for PHF tau disease. It effectively reduced the ApoE-dependent induction of the state (Figure 43H; ** p<0.01, *** p <0.001). As shown in Figures 43D and 43H, the PHF tau level is, It was significantly reduced by 1343Ah ( ** p<0.01, *** p<0.001).

[0307] The similarity in efficacy between mouse monoclonal antibodies and their corresponding humanized antibodies is similar to that of ApoE. Alternatively, the affinity and binding properties of the binding domain to ApoEch are maintained during humanization. This makes us confident that the binding properties of CDRs differ from those of the first mouse IgG1, and human I. This demonstrates that it can transfer to other proteins, including gG2.

[0308] The binding affinity between ApoE3 and monoclonal antibody 1H4 (mAb 1H4) is B Using the advanced dynamic module of the LItz system (Blitz Pro, FB-609928, ver. 1.3.1.3) And so the decision was made. To put it simply, the protein A biosensor was loaded with mAb 1H4. Both the association and dissociation constants are set for the full length ApoE3 tan from 0 nM to 571.4 nM. The increase in concentration of the protein (Innovagen) was determined. The following execution settings were used. : Initial baseline of 30 seconds in the experimental medium, ligand (mAb) on the biosensor 1H4) ​​120-second loading, association (120 seconds), and dissociation (120 seconds) steps New baseline for the previous 30 seconds. 420 seconds total at 2200 rpm and room temperature. A total of six runs were performed to determine the binding parameters of ApoE3. The overall results of the dissociation experiment Fitting and step correction are performed using BLitz software (ver. 1.1.0.7) The procedure was performed using ). Figure 44 shows the increase in concentration (nM) on the protein A biosensor. Representative binding measurements of the ApoE3 protein with 1H4 are shown. KD, Ka, and Kd The following parameters were measured (Table 10) and calculated using the BLitz system. The upper panel shows the coupling dynamics. The panel below represents the association step, and the panel below represents the dissociation step. The X and Y axes are... These show the time in seconds and the bond in nM, respectively.

[0309] [Table 13]

[0310] [Table 14]

[0311] [Example 10] In vivo validation of APOE fragment fusion proteins Next, it contains an APOE fragment containing an HSPG-binding domain and the Fc region of human IgG. The resulting fusion protein was tested using a similar in vivo model. In short, 0 Tau pathology was induced using 0.78 μg of recombinant full-length APOE. The protein was injected into the vitreous humor of mice, as shown in Figures 43E and 43I. Furthermore, the fusion protein reduced ApoE-dependent tau pathology in neurons.

[0312] [Example 11] Chimeric antibody sequence The sequence of the chimeric antibody in which the CDR was transferred from mouse to human IgG2 or IgG4 is as follows: As shown: Standard font = vector Italics = signal peptide Underline=VL / VH Double underline = steady state (human kappa / IgG4 / IgG2) Expression vector sequence for mAb 1H4 IgG2 / kappa: >p1H4.VL.hk

[0313] [ka]

[0314] [ka] >p1H4.VH.hIgG2

[0315] [ka]

[0316] [ka]

[0317] [ka] Expression vector sequence for mAb 1343Ah IgG2 / kappa: >p1343Ah.VL.hk

[0318] [ka]

[0319] [ka] >p1343Ah.VH.hIgG2

[0320] [ka]

[0321] [ka]

[0322] [ka] Sequence of the expression vector for mAb 19G10 IgG4 / kappa: >p19G10.VL.hk

[0323] [ka]

[0324] [ka] >p19G10.VH.hIgG4

[0325] [ka]

[0326] [ka]

[0327] [ka] Sequence of the expression vector for mAb 25F1 IgG4 / kappa: >p25F1.VL.hk

[0328] [ka]

[0329] [ka] >p20F1.VH.hIgG4

[0330] [ka]

[0331] [ka]

[0332] [ka] Sequence of the expression vector for mAb 7C11.1 IgG2 / kappa: >p7C11.1.VL.hk

[0333] [ka]

[0334] [ka] >p7C11.1.VH.hIgG2

[0335] [ka]

[0336] [ka]

[0337] [ka]

[0338] References:

[0339] [Table 15-1]

[0340] [Table 15-2]

[0341] [Table 15-3]

[0342] [Table 15-4]

[0343] [Table 15-5]

[0344] Other Embodiments Although the present invention is described in detail, the foregoing description is intended to illustrate, not to limit, the scope of the invention, and it should be understood that the scope of the invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Various embodiments of the present invention are shown below. 1. An isolated monoclonal antibody that specifically binds to one or more HSPG binding sites of wild-type or mutant apolipoprotein E (ApoE), or to one or more sites of allosteric regulation of HSPG binding. 2. The antibody described in 1 above, which binds to a polypeptide having at least 95% the same amino acid sequence as TEELRVRLASHLRK (SEQ ID NO: 3). 3. The antibody described in 1 above, which binds to a polypeptide having at least 95% the same amino acid sequence as TEELRVSLASHLRK (SEQ ID NO: 2). 4. An antibody according to any one of 1 to 3 above, which binds to one or more HSPG binding sites of wild-type or mutant ApoE2, ApoE3, or ApoE4. 5. The antibody competes with and / or binds to the same epitope as the reference anti-ApoE antibody, which includes the heavy chain variable region (VH) and the light chain variable region (VL), and the VH and VL of the reference antibody are (i) The amino acid sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 12, respectively; (ii) The amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 22, respectively; (iii) the amino acid sequence shown in SEQ ID NO: 33 and the amino acid sequence shown in SEQ ID NO: 32, respectively; or (iv) The amino acid sequence shown in SEQ ID NO: 43 and the amino acid sequence shown in SEQ ID NO: 42, respectively The antibody described in any of items 1 to 4 above, including the antibody described in item 1 to 4 above. 6. The antibody according to any one of 1 to 4 above, wherein the antibody competes with and / or binds to the same epitope as a reference anti-ApoE antibody, which includes a heavy chain and a light chain, and the heavy chain and light chain of the reference antibody include the amino acid sequence shown in SEQ ID NO: 53 and the amino acid sequence shown in SEQ ID NO: 52. 7. Anti-ApoE antibodies comprising VH, which includes VHCDR1, VHCDR2, and VHCDR3, and VL, which includes VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are (i) respective sequence numbers 7, 8, 9, 4, 5, and 6; (ii) Sequence numbers 17, 18, 19, 14, 15, and 16, respectively; (iii) Sequence numbers 27, 28, 29, 24, 25, and 26, respectively; (iv) Sequence numbers 37, 38, 39, 34, 35, 36, respectively; or (v) Sequence IDs 47, 48, 49, 44, 45, and 46 respectively Antibodies containing antibodies. 8.(i)VH and VL contain amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 13 and 12, respectively; (ii) VH and VL contain amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs. 23 and 22, respectively; (iii) VH and VL contain amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 33 and 32, respectively; or (iv) VH and VL contain amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 43 and 42, respectively. The antibody described in item 7 above. 9. The antibody according to item 7 above, comprising a heavy chain and a light chain, each containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences shown in SEQ ID NOs. 53 and 52. 10. An antibody according to any one of items 1 to 9 above, comprising mouse IgG1, IgG2a, IgG2b, IgG2c, or the IgG triple heavy chain constant region. 11. An antibody according to any one of items 1 to 9 above, comprising the human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. 12. An antibody according to any of 1 to 11 above, comprising a human kappa or human lambda light chain constant region. 13. An antibody as described in any of items 1 to 12 above, which is a whole antibody, a single-domain antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, Fv, scFv, sc(Fv)2, a diabody, a nanobody, Fab, or F(ab')2. 14. An antibody according to any of items 1 to 13 above, further comprising a portion that extends the half-life. 15. An antibody according to any one of items 1 to 14 above, further comprising a blood-brain barrier permeable portion. 16. An antibody according to any one of items 1 to 12 above, further comprising a detectable label. 17. A pharmaceutical composition containing an antibody as described in any of items 1 to 16 above. 18. A polynucleotide encoding one of the antibodies described in any of items 1 to 13 above. 19. A vector containing the polynucleotide described in item 18 above. 20. A host cell containing the polynucleotide described in item 18 above or the vector described in item 19 above. 21. A method for producing an anti-ApoE antibody, (a) the step of culturing the host cells described in 20 above under conditions that enable the expression of the antibody; and (b) Step of isolating the antibody Methods that include... 22. The method according to 21, further comprising the step of formulating an antibody as a sterile preparation suitable for administration to humans. 23.STEELRVRLASHLRKLRKRLLRDADDLQK (Sequence ID 57), STEELRVSLASHLRKLRKRLLRDADDLQK (Sequence ID 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (Sequence ID 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL(Sequence ID 60) An Fc fusion protein containing an HSPG-binding domain of wild-type ApoE or mutant ApoE, which contains an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the following. 24. An Fc fusion protein as described in 23 above, comprising the Fc region of a human antibody. 25. The Fc fusion protein according to 24 above, wherein the human antibody is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 molecules. 26. A pharmaceutical composition containing the Fc fusion protein described in any of items 23 to 25 above. 27. A polynucleotide encoding the Fc fusion protein described in any of items 23-25 ​​above. 28. A vector containing the polynucleotide described in item 27 above. 29. A host cell containing the polynucleotide described in 28 above or the vector described in 28 above. 30.(i) HSPG-binding domains of wild-type ApoE or mutant ApoE containing an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60); and (ii) Pharmaceutically acceptable adjuvants A pharmaceutical composition for inducing an immune response, including [the specified element]. 31. A method for improving, slowing, delaying, preventing, or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need, comprising the step of administering a therapeutically effective amount of an antibody described in any of items 1 to 16 above, an Fc fusion protein described in any of items 23 to 25 above, or a pharmaceutical composition described in any of items 17 to 26 above to a human subject. 32. A method for improving, slowing, delaying, preventing, or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need, wherein the method is (i) A viral vector containing a nucleotide sequence encoding a gRNA molecule that includes a target domain and a targeting domain complementary to the APOE gene; (ii) Viral vectors containing nucleotide sequences encoding the Cas9 molecule; and (iii) A viral vector containing a template nucleic acid that includes adenine:g.45412013C>A in place of cytosine at position 19 of the APOE gene. A method comprising the step of administering to the subject, wherein the administration results in the generation of one or more ApoE R136S alleles in one or more cells of the subject. 33. The method according to 32 above, wherein the targeting domain of the gRNA molecule is the same as the sequence from Table 7, or contains a sequence that differs from it by only 3 nucleotides or less. 34. The method described in 31 or 32 above, wherein the human subject has been diagnosed with Alzheimer's disease or is at risk of developing it. 35. The method according to 34 above, wherein the human subject has one or more copies of the APOE4 allele. 36. The method according to 34 above, wherein the human subject has one or more mutations in at least one gene selected from the group consisting of APP, PSEN1, and PSEN2. 37. The method according to 34 above, wherein the human subject has all or part of the third copy of chromosome 21. 38. The method described in 34 above, wherein the human subject has been diagnosed with Alzheimer's disease by brain imaging. 39. The method described in 34 above, wherein the human subject is over 50 years old. 40. Human subjects diagnosed with or at risk of developing a disorder selected from the following: vascular cognitive impairment, vascular dementia, autosomal overt cerebral artery disease with subcortical infarction and leukoencephalopathy (CADASIL), autosomal claustrophobic cerebral artery disease with subcortical infarction and leukoencephalopathy (CARASIL), Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease, neurodegenerative diseases, cerebrovascular diseases, brain injury, chronic traumatic brain injury, tauopathy, amyloidopathy, synucleinopathy, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, and aging. 41. A method for identifying human subjects less likely to develop early-onset neurodegenerative diseases, including the following steps: Obtaining a biological sample from the aforementioned subject, or the step of obtaining one; A step of detecting the presence of at least one mutant allele of APOE3 or the presence of a mutant ApoE3 gene product in the biological sample; and A step of identifying subjects who are less likely to develop early-onset neurodegenerative diseases based on the presence of a mutant ApoE3 allele or gene product in the aforementioned biological sample. 42. The method according to 41 above, wherein the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous fluid, aqueous humor, or tissue sample. 43. The method according to 41 above, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, retinal degeneration, or glaucoma. 44. The method according to 43 above, wherein the retinal degeneration is age-related macular degeneration. 45. The method according to 41, wherein the detection step includes determining the sequence of the APOE3 allele in the subject. 46. ​​The method according to 41, wherein the detection step includes determining the presence or absence of an APOE3 sequence encoding an ApoE3 protein having a mutation at R136, compared to a wild-type ApoE3 protein. 47. The method according to 46 above, wherein the mutation in R136 is R136S, R136H, or R136C. 48. The method according to any one of the above 41 to 47, further comprising the steps of selecting subjects for inclusion in a clinical trial, and optionally, performing experimental treatment, or, if the subject does not have a variant APOE3 allele, excluding the subject from the clinical trial. 49. The method according to any one of the above 41 to 47, further comprising the steps of selecting subjects for inclusion in a clinical trial, and optionally, performing experimental treatment, or, if the subject has a variant APOE3 allele, excluding the subject from the clinical trial.

Claims

1. An Fc fusion protein comprising an Fc region of an antibody fused to a polypeptide containing an HSPG-binding domain of wild-type ApoE or mutant ApoE, wherein the HSPG-binding domain comprises the amino acid sequence described in SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO:

60.

2. The Fc fusion protein according to claim 1, comprising the Fc region of a human antibody.

3. The Fc fusion protein according to claim 2, wherein the human antibody is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 molecules.

4. The Fc fusion protein according to any one of claims 1 to 3, wherein the Fc fusion protein comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

5. An Fc fusion protein according to any one of claims 1 to 4, comprising a human kappa or human lambda light chain constant region.

6. The Fc fusion protein according to any one of claims 1 to 5, further comprising a portion that extends the half-life.

7. The Fc fusion protein according to any one of claims 1 to 6, further comprising a blood-brain barrier permeable portion.

8. The Fc fusion protein according to any one of claims 1 to 7, further comprising a detectable label.

9. A pharmaceutical composition comprising the Fc fusion protein according to any one of claims 1 to 8.

10. Polynucleotide encoding the Fc fusion protein according to any one of claims 1 to 8 Ochido.

11. A vector comprising the polynucleotide described in claim 10.

12. A host cell comprising the polynucleotide according to claim 10, or the vector according to claim 11.

13. A method for producing an Fc fusion protein, (a) the step of culturing the host cells according to claim 12 under conditions that enable the expression of the Fc fusion protein; and (b) Step of isolating the Fc fusion protein Methods that include...

14. The method according to claim 13, further comprising the step of formulating an Fc fusion protein as a sterile formulation suitable for administration to humans.

15. A composition comprising an Fc fusion protein according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 9, for improving, slowing, delaying, preventing, or reversing dementia, mild cognitive impairment, and / or age-related cognitive decline in a human subject in need.

16. The composition or pharmaceutical composition according to claim 15, wherein the human subject has been diagnosed with Alzheimer's disease or is at risk of developing it.

17. The composition or pharmaceutical composition according to claim 15 or 16, wherein the human subject has one or more copies of the APOE4 allele.

18. The composition or pharmaceutical composition according to any one of claims 15 to 17, wherein the human subject has one or more mutations in at least one gene selected from the group consisting of APP, PSEN1, and PSEN2.

19. The composition or pharmaceutical composition according to any one of claims 15 to 18, wherein the human subject has all or part of the third copy of chromosome 21.

20. The composition or pharmaceutical composition according to any one of claims 15 to 19, wherein the human subject has been diagnosed with Alzheimer's disease by brain imaging.

21. A composition or pharmaceutical composition according to any one of claims 15 to 20, wherein the human subject is over 50 years of age.

22. A composition or pharmaceutical composition according to any one of claims 15 to 21, wherein a human subject has been diagnosed with or is at risk of developing a disorder selected from the group consisting of the following. Vascular cognitive impairment, vascular dementia, autosomal overt cerebral artery disease with subcortical infarction and leukoencephalopathy (CADASIL), autosomal claustrophobic cerebral artery disease with subcortical infarction and leukoencephalopathy (CARASIL), Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease, neurodegenerative diseases, cerebrovascular diseases, brain injury, chronic traumatic brain injury, tauopathy, amyloidopathy, synucleinopathy, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, age-related macular degeneration, and aging.

23. The composition or pharmaceutical composition according to any one of claims 15 to 22, wherein improving, slowing, delaying, preventing, or reversing dementia, mild cognitive impairment and / or age-related cognitive decline in a human subject in need comprises administering a therapeutically effective amount of the composition or pharmaceutical composition to a human subject.

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